U.S. patent application number 15/973715 was filed with the patent office on 2018-09-13 for expression system.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS S.A.. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS S.A.. Invention is credited to Normand BLAIS, Philippe Marc Helene DEHOTTAY, Marianne DEWERCHIN, Philippe GOFFIN, Denis MARTIN.
Application Number | 20180258146 15/973715 |
Document ID | / |
Family ID | 41402733 |
Filed Date | 2018-09-13 |
United States Patent
Application |
20180258146 |
Kind Code |
A1 |
BLAIS; Normand ; et
al. |
September 13, 2018 |
EXPRESSION SYSTEM
Abstract
Methods for producing a conjugate of a bacterial toxin,
including diphtheria toxin or CRM197, are provided.
Inventors: |
BLAIS; Normand; (Laval,
CA) ; DEHOTTAY; Philippe Marc Helene; (Rixensart,
BE) ; DEWERCHIN; Marianne; (Rixensart, BE) ;
GOFFIN; Philippe; (Rixensart, BE) ; MARTIN;
Denis; (Laval, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS S.A. |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
S.A.
Rixensart
BE
|
Family ID: |
41402733 |
Appl. No.: |
15/973715 |
Filed: |
May 8, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15210245 |
Jul 14, 2016 |
9994622 |
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15973715 |
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13500244 |
Apr 4, 2012 |
9422345 |
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PCT/EP2010/065047 |
Oct 7, 2010 |
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15210245 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/034 20130101;
C07K 1/00 20130101; A61P 31/04 20180101; C12Y 204/02036 20130101;
C07K 14/34 20130101; C12N 9/1077 20130101 |
International
Class: |
C07K 14/34 20060101
C07K014/34; C12N 9/10 20060101 C12N009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 8, 2009 |
GB |
0917647.0 |
Claims
1. A process for producing a conjugate of a bacterial toxin,
comprising: (1) growing a culture of Escherichia coli (E. coli)
host cells containing a polynucleotide comprising: (a) a 3' toxin
sequence encoding a mature bacterial toxin polypeptide having an
amino acid sequence at least 90% identical to SEQ ID NO:32, and (b)
a 5' signal sequence encoding a signal peptide, wherein the signal
peptide directs transport of said bacterial toxin polypeptide to
the bacterial periplasm when expressed in said host cell, and
wherein the 5' signal sequence is not derived from Corynebacterium
diphtheriae (C. diphtheriae); and inducing expression of said
polynucleotide such that said bacterial toxin polypeptide is
expressed periplasmically; and (2) harvesting cell paste from the
culture and purifying said bacterial toxin polypeptide; and (3)
conjugating said purified bacterial toxin to an antigen; wherein
the process of (1) is carried out in a fermentor.
2. The process of claim 1 wherein the 3' toxin sequence encodes a
polypeptide selected from the group consisting of (a) a polypeptide
having the amino acid sequence of SEQ ID NO: 32, (b) a polypeptide
having at least 95% sequence identity to SEQ ID NO: 32, (c) a
polypeptide comprising SEQ ID NO:31, and (d) CRM197.
3. The process of claim 1 wherein the polynucleotide encodes a
polypeptide comprising any one of SEQ ID NOs: 33-45.
4. The process of claim 1 wherein the 5' signal sequence is
directly 5' of the 3' toxin sequence of the polynucleotide.
5. The process of claim 1 wherein the 5' signal sequence encodes a
signal peptide having an amino acid sequence selected from: (a) SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, and (b)
variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
and 26 containing 1, 2 or 3 point mutations, insertions or
deletions, which variants are capable of directing transport of
said bacterial toxin polypeptide to the periplasm of said bacterial
host cell, and (c) fragments of at least 10 amino acids of SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26, which
fragments are capable of directing transport of said bacterial
toxin polypeptide to the periplasm of said bacterial host cell.
6. The process of claim 1 wherein the 5' signal sequence comprises
a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, and 25.
7. The process of claim 1 where said antigen of step (3) is a
capsular bacterial saccharide from a bacterium selected from the
group consisting of Streptococcus pneumoniae, Haemophilus
influenzae, Neisseria meningitidis, group B Streptococcus, group A
Streptococcus, Salmonella, Enterococci, and Staphylococcus
aureus.
8. The process of claim 7, where the activated saccharide is
conjugated to said bacterial toxin via a linker.
9. The process of claim 7, where conjugation of said purified
bacterial toxin to an antigen comprises a process selected from:
(a) direct reductive amination, (b) activation of the saccharide by
1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), (c)
activation of the saccharide by cyanogen bromide, (d) activation of
the saccharide by 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) followed by derivitisation of the
activated saccharide with adipic acid dihydrazide (ADH), and (e)
activation of the saccharide by cyanogen bromide followed by
derivitisation of the activated saccharide with ADH.
10. A process for producing a conjugate of a bacterial toxin,
comprising: (1) growing a culture of Escherichia coli (E. coli)
host cells containing a polynucleotide comprising: (a) a 3' toxin
sequence encoding a mature bacterial toxin polypeptide having an
amino acid sequence at least 90% identical to SEQ ID NO:32, and (b)
a 5' signal sequence encoding a signal peptide, wherein the signal
peptide directs transport of said bacterial toxin polypeptide to
the bacterial periplasm when expressed in said host cell, and
wherein the 5' signal sequence is not derived from C. diphtheriae,
and wherein the signal peptide has an amino acid sequence selected
from: (i) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
and 26, and (ii) variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, and 26 containing 1, 2 or 3 point mutations,
insertions or deletions, which variants are capable of directing
transport of said bacterial toxin polypeptide to the periplasm of
said bacterial host cell, and (iii) fragments of at least 10 amino
acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and
26, which fragments are capable of directing transport of said
bacterial toxin polypeptide to the periplasm of said bacterial host
cell; and inducing expression of said polynucleotide such that said
bacterial toxin polypeptide is expressed periplasmically; and (2)
harvesting cell paste from the culture and purifying said bacterial
toxin polypeptide; and (3) conjugating said purified bacterial
toxin to an antigen; wherein the process of (1) is carried out in a
fermentor.
11. The process of claim 10 wherein the 3' toxin sequence encodes
encodes a polypeptide selected from the group consisting of (a) a
polypeptide having the amino acid sequence of SEQ ID NO: 32, (b) a
polypeptide having at least 95% sequence identity to SEQ ID NO: 32,
(c) a polypeptide comprising SEQ ID NO:31, and (d) CRM197.
12. The process of claim 10 wherein the polynucleotide encodes a
polypeptide comprising any one of SEQ ID NOs: 33-45.
13. The process of claim 10 wherein the 5' signal sequence is
directly 5' of the 3' toxin sequence of the polynucleotide.
14. The process of claim 10, wherein the 5' signal sequence encodes
a signal peptide having an amino acid sequence selected from: (a)
SEQ ID NO: 24, and (b) variants of SEQ ID NO: 24, varying from the
corresponding sequence by 1, 2, or 3 point mutations, amino acid
insertions, or amino acid deletions, which variants are capable of
directing transport of said bacterial toxin polypeptide to the
periplasm of said bacterial host cell, and (c) fragments of at
least 10 amino acids of SEQ ID NO: 24, which fragments are capable
of directing transport of said bacterial toxin polypeptide to the
periplasm of said bacterial host cell.
15. The process of claim 10 wherein the 5' signal sequence
comprises a nucleic acid sequence selected from SEQ ID NO: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, and 25.
16. The process of claim 10 where said antigen of step (3) is a
capsular bacterial saccharide from a bacterium selected from the
group consisting of Streptococcus pneumoniae, Haemophilus
influenzae, Neisseria meningitidis, group B Streptococcus, group A
Streptococcus, Salmonella, Enterococci, and Staphylococcus
aureus.
17. The process of claim 16, where the activated saccharide is
conjugated to said bacterial toxin via a linker.
18. The process of claim 16, where conjugation of said purified
bacterial toxin to an antigen comprises a process selected from:
(a) direct reductive amination, (b) activation of the saccharide by
1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP), (c)
activation of the saccharide by cyanogen bromide, (d) activation of
the saccharide by 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) followed by derivitisation of the
activated saccharide with adipic acid dihydrazide (ADH), and (e)
activation of the saccharide by cyanogen bromide followed by
derivitisation of the activated saccharide with ADH.
19. A process for manufacturing an immunogenic composition
comprising the steps of: (a) producing a conjugate of a bacterial
toxin according to claim 1; and (b) mixing the said conjugate with
a component selected from a pharmaceutically acceptable excipient,
a further antigen, and an adjuvant.
20. A process for manufacturing an immunogenic composition
comprising the steps of: (a) producing a conjugate of a bacterial
toxin according to claim 16; and (b) mixing the said conjugate with
a component selected from a pharmaceutically acceptable excipient,
a further antigen, and an adjuvant.
Description
[0001] This application is a Continuation application of U.S.
Continuation application Ser. No. 15/210,245 filed Jul. 14, 2016,
allowed, which is a continuation of U.S. patent application Ser.
No. 13/500,244 filed Apr. 4, 2012, now U.S. Pat. No. 9,422,345,
issued Aug. 23, 2016, which was filed pursuant to 35 U.S.C. .sctn.
371 as a U.S. National Phase Application of International Patent
Application Serial No. PCT/EP2010/065047 filed Oct. 7, 2010, which
claims priority to United Kingdom Patent Application No.
GB0917646.0 filed Oct. 8, 2009, and the entire contents of each of
the foregoing applications are hereby incorporated by
reference.
DESCRIPTION
[0002] The present invention relates to the field of the expression
of bacterial toxins, in particular diphtheria toxins (including
mutant forms of diphtheria toxin, such as CRM197) and includes
processes suitable for the expression and manufacture of bulk
cultures of such toxins. The invention also provides novel
polynucleotides and polypeptides which can be used or produced
during the processes of the invention.
[0003] Diphtheria toxin is a protein exotoxin produced by the
bacterium Corynebacterium diphtheria. It is produced as a single
polypeptide containing a signal sequence (the tox signal sequence)
that is removed by the bacterium on secretion of the protein.
[0004] The diphtheria toxin is readily spliced to form two subunits
linked by a disulphide bond, Fragment A and Fragment B, as a result
of cleavage at residue 190, 192 or 193 (Moskaug et al Biol. Chem.
264: 15709-15713, 1989). Fragment A is the catalytically active
portion and is an NAD-dependent ADP-ribosyltransferase which
specifically targets a protein synthesis factor EF-2, thereby
inactivating EF-2 and shutting down protein synthesis in a
cell.
[0005] Immunity to a bacterial toxin such as diphtheria toxin may
be acquired naturally during the course of infection, or
artificially by injection of a detoxified form of the toxin
(toxoid) (Germanier, Bacterial Vaccines, Academic Press, Orlando,
Fl., 1984). Toxoids have traditionally been made by chemical
modification of native toxins (Lingood et al Brit. J. Exp. Path.
44; 177, 1963), rendering them non-toxic while retaining an
antigenicity that protects the vaccinated animal against subsequent
challenge by the natural toxin. Alternatively, several mutated
diphtheria toxins have been described which have reduced toxicity
(U.S. Pat. No. 4,709,017, U.S. Pat. No. 4,950,740).
[0006] CRM197 is a non-toxic form of the diphtheria toxin but is
immunologically indistinguishable from the diphtheria toxin. CRM197
is produced by C. diphtheriae infected by the nontoxigenic phase
.beta.197tox--created by nitrosoguanidine mutagenesis of the
toxigenic carynephage b (Uchida et al Nature New Biology (1971)
233; 8-11). The CRM197 protein has the same molecular weight as the
diphtheria toxin but differs from it by a single base change in the
structural gene. This leads to a glycine to glutamine change of
amino acid at position 52 which makes fragment A unable to bind NAD
and therefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46;
69-94, Rappuoli Applied and Environmental Microbiology September
1983 p 560-564).
[0007] Diphtheria toxoid and a mutant form with reduced toxicity,
CRM197, are components in many vaccines providing immunity against
Corynebacterium diphtheriae. Several combination vaccines are known
which can prevent Bordetella pertussis, Clostridium tetani,
Corynebacterium diphtheriae, and optionally Hepatitis B virus
and/or Haemophilus influenzae type b (see, for instance, WO
93/24148 and WO 97/00697, WO 02/055105).
[0008] Diphtheria toxin and mutant forms including CRM197 have also
been used in vaccines as safe and effective T-cell dependent
carriers for saccharides. CRM197 is currently used in the
Haemophilus influenzae type b oligosaccharide CRM197 conjugate
vaccine (HibTitre.RTM.; Lederle Praxis Biologicals, Rochester,
N.Y.).
[0009] Methods of preparing diphtheria toxoid (DT) are well known
in the art. For instance, DT may be produced by purification of the
toxin from a culture of Corynebacterium diphtheriae followed by
chemical detoxification, or may be made by purification of a
recombinant, or genetically detoxified analogue of the toxin (for
example, CRM197, or other mutants as described in U.S. Pat. No.
4,709,017, U.S. Pat. No. 5,843,711, U.S. Pat. No. 5,601,827, and
U.S. Pat. No. 5,917,017).
[0010] Production of significant quantities of diphtheria toxins
such as CRM197 for use in vaccines has been hindered due to low
protein abundance. This problem has been addressed previously by
expressing CRM197 in E. coli (Bishai et al J Bacteriol.
169:5140-5151), Bishai et al describes the expression of a
recombinant fusion protein containing diphtheria toxin (including
the tox signal sequence) this led to the production of degraded
protein.
[0011] Cloning of Diptheria fragments containing the tox signal
sequence and expression of these sequences in Escherichia coli
involves certain difficulties. The expressed protein is secreted
into the periplasmic space and this secretion is associated with
decreased viability of the host cells (O'Keefe et al Proc. Natl.
Acad. Sci. 86:343-346) and increased proteolysis of the recombinant
protein (Bishai et al J Bacteriol. 169: 5140-5151). For these
reasons removal of the tox signal sequence so that expression is no
longer periplasmic has been suggested, this can increase expression
of Diphtheria toxoids (Bishai et al).
[0012] Accordingly, the present application provides an improved
process for making a bacterial toxin by periplasmic expression
comprising the steps of a) growing a culture of the bacterial host
cell containing an expression vector in which particular signal
sequences are linked to the sequence of a bacterial toxin and b)
inducing expression of the polypeptide containing particular signal
sequences linked to a bacterial toxin such that a bacterial toxin
is expressed periplasmically. The present application also provides
polynucleotides which are used in the process of the invention.
[0013] Production of bacterial toxins in the periplasm may have one
or more advantages over cytoplasmic production. [0014] (1) The
protein is produced in its mature form after cleavage of the signal
peptide, and/or; [0015] (2) The periplasm of E. coli is an
oxidizing environment that allows the formation of disulphide
bonds, this may help produce soluble, correctly folded proteins,
and/or; [0016] (3) The periplasm of E. coli contains fewer
proteases than the cytoplasm, this may help avoid proteolytic
cleavage of the expressed protein, and/or; [0017] (4) The periplasm
also contains fewer proteins, this allows purer recombinant protein
to be obtained.
SUMMARY OF THE INVENTION
[0018] In a first aspect of the invention there is provided a
polynucleotide comprising a 5' signal sequence portion and a 3'
toxin portion wherein; [0019] (a) The 5' signal sequence portion
encodes a heterologous polypeptide having an amino acid sequence
capable of directing transport of a heterologous protein to the
bacterial periplasm; and [0020] (b) the 3' toxin portion encodes a
polypeptide having an amino acid sequence at least 90% identical to
SEQ ID NO: 32 or fragments thereof encoding at least 15 amino acids
and/or at least one B or T cell epitope.
[0021] In a second aspect of the invention there is provided a
polynucleotide comprising a 5' signal sequence portion and a 3'
toxin portion wherein [0022] (a) the 5' signal sequence portion
encodes a polypeptide having an amino acid sequence capable of
directing transport of a heterologous protein to the bacterial
periplasm and wherein the 5' signal sequence is not derived from C.
diphtheriae; and [0023] (b) the 3' toxin portion encodes a
polypeptide having an amino acid sequence at least 90% identical to
SEQ ID NO: 32 or fragments thereof encoding at least 15 amino acids
and/or at least one B or T cell epitope.
[0024] In a third aspect of the invention there is provided a
polynucleotide comprising a 5' signal sequence portion and a 3'
toxin portion wherein the 5' signal portion sequence encodes a
signal peptide having an amino acid sequence of [0025] (a) SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; [0026] (b)
variants of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
or 26, varying from the corresponding sequences by 1, 2 or 3 point
mutations, amino acid insertions or amino acid deletions, which are
capable of directing an expressed protein to the periplasm; or
[0027] (c) fragments of at least 10 amino acids of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 which are capable of
directing an expressed protein to the periplasm; [0028] and the 3'
toxin portion encodes a bacterial toxin or fragment or variant
thereof.
[0029] In a fourth aspect of the invention there is provided a
vector comprising the polynucleotide of the invention linked to an
inducible promoter.
[0030] In a fifth aspect of the invention there is provided a host
cell comprising the polynucleotide or the vector of the
invention.
[0031] In a sixth aspect of the invention there is provided a
polypeptide encoded by the polynucleotide of the invention.
[0032] In a seventh aspect of the invention there is provided a
process for making a bacterial toxin comprising the steps of a)
growing a culture of the bacterial host cell of the invention and
b) inducing expression of the polypeptide such that a bacterial
toxoid is expressed periplasmically.
[0033] In an eighth aspect of the invention there is provided a
process for making a conjugate comprising the steps of a) making a
bacterial toxin using the process of the invention and b)
conjugating the bacterial toxin of step a) to an antigen.
[0034] In a ninth aspect of the invention there is provided a
process for manufacturing a vaccine comprising the steps of a)
making a bacterial toxin or conjugate using the process of the
invention and b) mixing the bacterial toxin or conjugate thereof
with a pharmaceutically acceptable excipient.
DESCRIPTION OF THE FIGURES
[0035] FIG. 1--Overview of the cloning process to produce the
CRM197-signal sequence constructs. A section of DNA encoding a
signal sequence fused to the N-terminus of CRM197 is cut from
plasmid A by NdeI and SacI digestion. The C-terminal sequence of
CRM197 SEQ ID NO:31 is cut from plasmid B by AatII and XhoI
digestion. These two sequences are spliced into a pET26b vector by
digestion of the pET26b vector with NdeI, SacI, AatII and XhoI and
ligation using DNA ligase. The resulting vector (pRIT16668) was
used to produce the other signal sequence-CRM197 constructs by
digesting plasmids containing the signal sequences and the
pRIT16668 vector with AgeI and NdeI and splicing these together
using DNA ligase.
[0036] FIG. 2--Overview of the cloning process to produce
constructs containing the CRM197 sequence without an N-terminal
signal sequence (pRIT16669). This was carried out by inserting a
PCR fragment encoding CRM197 into pRIT16668 using NdeI and AatII
digestion followed by ligation.
[0037] FIG. 3--Overview of the cloning process to produce
constructs containing the FlgI signal sequence. This was carried
out by inserting a PCR fragment encoding the CRM197 and the FlgI
signal sequence (SEQ ID NO: 23) into pRIT16669 using NdeI and AatII
digestion followed by ligation. This produced the plasmid
pRIT16681.
[0038] FIG. 4--Gels demonstrating induction of DsbA-CRM197 3 hours
after treatment with IPTG at 30.degree. C. in three different
strains. Novex TG 10-20% gel was used. Panel A gel is a Western
blot stained using anti-DTPa and anti-mouse NBT-BCIP. Panel B gel
is stained using Coomassie blue. Lane 1 contains the molecular
weight marker, lane 2 contains a BLR(DE3) strain which has not been
induced, lane 3 contains a BLR(DE3) strain which has been induced,
lane 4 contains a B834 (DE3) strain which has not been induced,
lane 5 contains a B384 (DE3) strain which has been induced and lane
6 contains an HMS174(DE3) strain which has been induced.
[0039] FIG. 5--Gels demonstrating expression of CRM-197 in the
bacterial cell and in the culture medium. Panel B gel shows a
Western blot, stained using anti-DTPa and anti-mouse NBT-BCIP.
Panel A is stained using Coomassie blue. The contents of each lane
are described in the table below:
TABLE-US-00001 TABLE 1 Lane Signal SEQ ID NO SEQ ID NO Number
sequence Culture temperature nucleotide Amino acid 1 DsbA Overnight
23.degree. C. 25 26 2 OmpA Overnight 23.degree. C. 5 6 3 NspA
Overnight 23.degree. C. 7 8 4 FlgI Overnight 23.degree. C. 23 24 5
TolB Overnight 23.degree. C. 19 20 6 SfmC Overnight 23.degree. C.
11 12 7 TorT Overnight 23.degree. C. 9 10 8 FocC Overnight
23.degree. C. 13 14 9 Ccmh Overnight 23.degree. C. 15 16 10 Yra1
Overnight 23.degree. C. 17 18 11 NikA Overnight 23.degree. C. 21 22
12 PhtE Overnight 23.degree. C. 1 2 13 SipA Overnight 23.degree. C.
3 4 14 Marker 250, 150, 100 75, 50, 37, 25, 20 15 DsbA Non induced
25 26 16 DsbA Overnight 30.degree. C. 25 26 17 FocC Overnight
30.degree. C. 13 14 18 CcmH Overnight 30.degree. C. 15 16
[0040] FIG. 6--Gels demonstrating induction of DsbA-CRM197 for 3
hours at 30.degree. C. in three different strains. Novex TG 10-20%
gel was used. Panel A is a Western blot stained using anti-DTPa and
anti-mouse NBT-BCIP. Panel B is stained using Coomassie blue. Lane
1 contains the molecular weight marker, Lane 2 contains the
BLR(DE3) strain which has not been induced, lane 3 contains the
BLR(DE3) strain which has been induced, lane 4 contains the soluble
fraction from a BLR(DE3) strain that has been induced, lane 5
contains the insoluble fraction from a BLR(DE3) strain that has
been induced, lane 6 contains the B834(DE3) strain which has not
been induced, lane 7 contains the B834(DE3) induced soluble
fraction and lane 8 contains the B834(DE3) induced insoluble
fraction.
[0041] FIG. 7--Gels comparing the expression of the optimised FlgI
construct with or without the addition of 1M potassium phosphate
buffer to a concentration of 100 mM in the induction phase. Novex
TG 10-20% gel was used. Lane 1 contains the molecular weight
marker, lane 2 contains the cell extract at 0 minutes from
induction (induction at 30.degree. C.), lane 3 contains cell
extract at 2 hours from induction (induction at 30.degree. C.),
lane 4 contains cell extract at 4 hours from induction (induction
at 30.degree. C.), lane 5 contains cell extract after overnight
induction at 30.degree. C., lane 6 contains the medium after 4
hours after induction at 30.degree. C., lane 7 contains the medium
over after induction at 30.degree. C. overnight, lane 8 contains
cell extract at two hours from induction (induction at 23.degree.
C.), lane 9 contains cell extract at 4 hours from induction
(induction at 23.degree. C.), lane 10 contains cell extract after
overnight induction, lane 11 contains medium 4 hours from induction
(induction at 23.degree. C.) and lane 12 contains medium after
overnight induction (induction at 23.degree. C.).
[0042] FIG. 8--Depiction of a fermentation profile with the process
parameters monitored during 20 litre scale fed-batch fermentation.
Line 1 describes the amount of substrate added (grams), line 2
describes the pH, line 3 describes the stirring rate (rpm), line 4
describes the pO.sub.2 (%), line 5 describes the temperature
(.degree. C.) and line 6 describes the amount of base added
(grams).
[0043] FIG. 9A-9G--Sequence listings of polypeptides and
polynucleotides of the invention.
[0044] FIG. 9A provides SEQ ID NOs: 1-9;
[0045] FIG. 9B provides SEQ ID NOs: 10-18;
[0046] FIG. 9C provides SEQ ID NOs: 19-27;
[0047] FIG. 9D provides SEQ ID NOs: 28-31;
[0048] FIG. 9E provides SEQ ID NOs: 32-34;
[0049] FIG. 9F provides SEQ ID NOs: 35-40;
[0050] FIG. 9G provides SEQ ID NOs: 41-45.
[0051] FIG. 10--Depiction of the production of CRM197 in the
periplasmic and cell-associated fractions as a function of the feed
rate and pH during induction, for growth performed at pH 6.8. The
left panel (A) shows periplasmic CRM197 production. The right panel
(B) describes cell-associated CRM197 production.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0052] The term "Polynucleotide(s)" generally refers to any
polyribonucleotide or polydeoxyribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA including single and
double-stranded regions/forms.
[0053] The term polypeptide refers to any peptide comprising at
least ten amino acids.
[0054] The term "polynucleotide encoding a peptide" as used herein
encompasses polynucleotides that include a sequence encoding a
peptide or polypeptide of the invention. The term also encompasses
polynucleotides that include a single continuous region or
discontinuous regions encoding the peptide or polypeptide (for
example, polynucleotides interrupted by integrated phage, an
integrated insertion sequence, an integrated vector sequence, an
integrated transposon sequence, or due to RNA editing or genomic
DNA reorganization) together with additional regions, that also may
contain coding and/or non-coding sequences.
[0055] Polynucleotide "variants of the 5' signal sequence portion"
are polynucleotide sequences encoding signal sequences which
contain, 1, 2, 3, 4 or 5 amino acid substitution, amino acid
addition or amino acid deletion mutations compared to the
corresponding wild type signal sequence.
[0056] An "amino acid deletion" is the removal of one amino acid
from the amino acid sequence of a protein.
[0057] An "amino acid addition" is the addition of one amino acid
from the amino acid sequence of a protein.
[0058] An "amino acid substitution is the replacement of one amino
acid with another amino acid, in the sequence of a protein.
[0059] Polynucleotide "variants of the 3' toxin portion" are
polynucleotide sequences which encode polypeptide sequences having
80%, 85%, 90%, 95%, 98% or 100% identity to a toxin polypeptide. A
definition for identity is given below.
[0060] In general "variants" are optionally polypeptides that vary
from the referents by conservative amino acid substitutions,
whereby a residue is substituted by another with like
characteristics. Typically such substitutions are among Ala, Val,
Leu and Ile; among Ser and Thr; among the acidic residues Asp and
Glu; among Asn and Gln; and among the basic residues Lys and Arg;
or aromatic residues Phe and Tyr.
[0061] "Fragments of the 5' signal sequence portion" are sequences
which encode at least 10, 15 or 20 amino acids of a signal
peptide.
[0062] "Fragments of the 3' toxin portion" are sequences which
encode a contiguous portion of at least 5, 10, 15, 20, 30, 40, 50,
100 or 200 amino acids of a toxin polypeptide, which optionally
have immunogenic activity. A peptide with "immunogenic activity" or
which is "immunogenic" is capable (if necessary when coupled to a
carrier) of raising an immune response which recognises the
respective toxin. Preferred fragments are those polynucleotides
which encode a B-cell or T-cell epitope, and recombinant,
polynucleotides comprising said polynucleotide fragments.
Optionally fragments of these toxins are fragments which are
immunogenic.
[0063] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences or two or more polynucleotide
sequences, as the case may be, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" can be readily calculated by known methods,
including but not limited to those described in (Computational
Molecular Biology, Lesk, A. M., ed., Oxford University Press, New
York, 1988; Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York, 1993; Computer Analysis of
Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds.,
Humana Press, New Jersey, 1994; Sequence Analysis in Molecular
Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis
Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New
York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,
48: 1073 (1988). Methods to determine identity are designed to give
the largest match between the sequences tested. Moreover, methods
to determine identity are codified in publicly available computer
programs. Computer program methods to determine identity between
two sequences include, but are not limited to, the GAP program in
the GCG program package (Devereux, J., et al., Nucleic Acids
Research 12(1): 387 (1984)), BLASTP, BLASTN (Altschul, S. F. et
al., J. Molec. Biol. 215: 403-410 (1990), and FASTA (Pearson and
Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST
family of programs is publicly available from NCBI and other
sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda,
Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
The well known Smith Waterman algorithm may also be used to
determine identity.
[0064] Parameters for polypeptide sequence comparison include the
following:
[0065] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0066] Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
[0067] Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)
[0068] Gap Penalty: 8
[0069] Gap Length Penalty: 2
[0070] A program useful with these parameters is publicly available
as the "gap" program from Genetics Computer Group, Madison Wis. The
aforementioned parameters are the default parameters for peptide
comparisons (along with no penalty for end gaps).
[0071] Parameters for polynucleotide comparison include the
following:
[0072] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453
(1970)
[0073] Comparison matrix: matches=+10, mismatch=0
[0074] Gap Penalty: 50
[0075] Gap Length Penalty: 3
[0076] Available as: The "gap" program from Genetics Computer
Group, Madison Wis. These are the default parameters for nucleic
acid comparisons.
[0077] The term `signal peptide` or `signal polypeptide` refers to
a peptide which is capable of directing an expressed protein to the
periplasm.
[0078] The term "bacterial toxin" encompasses both toxins and
toxoids.
[0079] The term "toxoid" describes toxins which have been partially
or completely inactivated by, for example the introduction of point
mutations, deletions or insertions.
[0080] The terms "comprising", "comprise" and "comprises" herein is
intended by the inventors to be optionally substitutable with the
terms "consisting of", "consist of", and "consists of",
respectively, in every instance.
[0081] The term `heterologous protein refers to a protein which is
not native to the cell type in which it is expressed. Similarly the
term `heterologous polypeptide` refers to a polypeptide which is
not native to the cell type in which it is expressed.
[0082] The term `polypeptide not derived from C. diphtheriae`
refers to a polypeptide which is different in sequence to a
polypeptide found in native (not recombinant) C. diphtheriae.
[0083] Polynucleotides of the Invention
[0084] One aspect of the invention relates to polynucleotides
encoding toxins which are periplasmically expressed.
[0085] In general the presence of a signal sequence on the protein
facilitates the transport of the protein into the periplasm
(prokaryotic hosts) or the secretion of the protein (eukaryotic
hosts). In prokaryotic hosts the signal sequence directs the
nascent protein across the inner membrane into the periplasmic
space; the signal sequence is then cleaved. A signal sequence is
capable of directing an expressed protein to the periplasm if, when
it is attached to a polypeptide of interest, during translation of
the polypeptide in a gram negative bacteria, more of said
polypeptide is found in the periplasm of a gram negative bacteria
than in the absence of the signal sequence. In an embodiment at
least 50, 60, 70, 80, 90 or 100% of the polypeptide of interest is
directed to the periplasm when expressed in a gram negative
bacterium such as E. coli.
[0086] An assay to test whether a signal sequence is capable of
directing periplasmic expression can be carried out using reporter
proteins. For example a periplasmic signal sequence can be spliced
upstream of a gene for a green fluorescent protein, this protein
can be expressed in a host cell of the invention. A microscope can
be used to judge the comparative levels of the green fluorescent
protein in the cytoplasm and the periplasm.
[0087] A protein or peptide is transported co-translationally if
transport occurs before the synthesis of a substantial amount of
the polypeptide chain. SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22,
24, or 26 encode peptides which are capable of directing an
expressed protein to the periplasm through co-translational
transport, whereas SEQ ID NOs: 6 and 8 encode peptides which are
capable of directing an expressed protein to the periplasm through
post-translational transport.
[0088] In an embodiment there is provided a polynucleotide (for
example for expression of a toxin in a bacterial cell) comprising a
5' signal sequence portion and a 3' toxin portion, wherein
[0089] The 5' signal sequence portion encodes a heterologous
(signal) polypeptide having an amino acid sequence capable of
directing transport of a heterologous (toxin) protein to the
bacterial periplasm; and [0090] (a) the 3' toxin portion encodes a
polypeptide having an amino acid sequence at least 75%, 80%, 85%,
90%, 95% or 99% identical to SEQ ID NO: 32 or fragments thereof
(which may be immunogenic) encoding at least 15 (contiguous) amino
acids and/or at least one B or T cell epitope.
[0091] In a further embodiment there is provided a polynucleotide
(for expression of a polypeptide in a bacterial cell) comprising a
5' signal sequence portion and a 3' toxin portion, wherein [0092]
(a) The 5' signal sequence portion encodes a polypeptide having an
amino acid sequence capable of directing transport of a
heterologous protein to the bacterial periplasm and wherein the 5'
signal sequence portion is not derived from C. diphtheriae; and
[0093] (b) the 3' toxin portion encodes a polypeptide having an
amino acid sequence at least 90% identical to SEQ ID NO: 32 or
fragments thereof encoding at least 15 amino acids and/or at least
one B or T cell epitope.
[0094] In one embodiment the 5' signal sequence is not the tox
signal sequence from Corynebacterium diphtheriae.
[0095] It is within the capabilities of the skilled person to
identify B and T cell epitopes. B cell epitopes may be identified
by 2D structure prediction, for example using the PSIPRED program
(from David Jones, Brunel Bioinformatics Group, Dept. Biological
Sciences, Brunel University, Uxbridge UB8 3PH, UK) (FIG. 4). The
antigenic index is calculated on the basis of the method described
by Jameson and Wolf (CABIOS 4:181-186 [1988]). The parameters used
in this program are the antigenic index and the minimal length for
an antigenic peptide. An antigenic index of 0.9 for a minimum of 5
consecutive amino acids was used as threshold in the program. T
cell epitopes may be identified, for example, by the tepitope
method describe by Sturniolo at al. (Nature Biotech. 17: 555-561
[1999]).
[0096] For clarity the phrase `having an amino acid sequence
capable of directing transport of a heterologous protein to the
bacterial periplasm` means the same as `having an amino acid
sequence capable of directing transport to the bacterial periplasm
of a heterologous protein`. In a further embodiment this
polynucleotide may encode a polypeptide having an amino acid
sequence capable of directing co-translational transport of a
heterologous protein to the bacterial periplasm.
[0097] Optionally the 3' toxin portion encodes SEQ ID NO 32.
Optionally the 3' toxin portion encodes DT. Optionally the 3' toxin
portion comprises SEQ ID NO: 31.
[0098] Optionally the 5' signal sequence portion encodes any one of
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or
any one of SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, 24, or 26,
or any one of SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, or 24,
or SEQ ID NO: 24 or any one of SEQ ID NO: 2, 4, or 24, or any one
of SEQ ID NO: 2, 10, or 24, or any one of SEQ ID NO: 2, 12, or 24,
or any one of SEQ ID NO: 2, 14, or 24, or any one of SEQ ID NO: 4,
10 or 24, or any one of SEQ ID NO: 4, 12, or 24, or any one of SEQ
ID NO: 4, 16, or 24, or any one of SEQ ID NO: 4, 18 or 24, or any
one of SEQ ID NO: 4, 20 or 24, or any one of SEQ ID NO: 4, 22, or
24, or any one of SEQ ID NO: 10, 12, or 24, or any one of SEQ ID
NO: 10, 14, or 24, or any one of SEQ ID NO: 10, 16, or 24, or any
one of SEQ ID NO: 10, 18, or 24, or any one of SEQ ID NO: 10, 22 or
24, or any one of SEQ ID NO: 12, 14, or 24, or any one of SEQ ID
NO: 12, 16, or 24, or any one of SEQ ID NO: 12, 18, or 24, or any
one of SEQ ID NO: 12, 20, or 24, or any one of SEQ ID NO: 12, 22,
or 24, or any one of SEQ ID NO: 14, 16, or 24, or any one of SEQ ID
NO: 14, 18, or 24, or any one of SEQ ID NO: 14, 20 or 24, or any
one of SEQ ID NO: 14, 22, or 24, or any one of SEQ ID NO: 16, 18,
or 24, or any one of SEQ ID NO: 16, 20 or 24, or any one of SEQ ID
NO: 16, 22, or 24, or any one of SEQ ID NO: 18, 20 or 24, or any
one of SEQ ID NO 18, 22, or 24.
[0099] In a further embodiment the 5' signal sequence portion
encodes (variants containing) 1, 2 or 3 point mutations, insertions
or deletions, of any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, or 26 or any one of SEQ ID NO: 2, 4, 10, 12, 14,
16, 18, 20, 22, 24, or 26, or any one of SEQ ID NO: 2, 4, 10, 12,
14, 16, 18, 20, 22, or 24, or SEQ ID NO: 24 or any one of SEQ ID
NO: 2, 4, or 24, or any one of SEQ ID NO: 2, 10, or 24, or any one
of SEQ ID NO: 2, 12, or 24, or any one of SEQ ID NO: 2, 14, or 24,
or any one of SEQ ID NO: 4, 10 or 24, or any one of SEQ ID NO: 4,
12, or 24, or any one of SEQ ID NO: 4, 16, or 24, or any one of SEQ
ID NO: 4, 18 or 24, or any one of SEQ ID NO: 4, 20 or 24, or any
one of SEQ ID NO: 4, 22, or 24, or any one of SEQ ID NO: 10, 12, or
24, or any one of SEQ ID NO: 10, 14, or 24, or any one of SEQ ID
NO: 10, 16, or 24, or any one of SEQ ID NO: 10, 18, or 24, or any
one of SEQ ID NO: 10, 22 or 24, or any one of SEQ ID NO: 12, 14, or
24, or any one of SEQ ID NO: 12, 16, or 24, or any one of SEQ ID
NO: 12, 18, or 24, or any one of SEQ ID NO: 12, 20, or 24, or any
one of SEQ ID NO: 12, 22, or 24, or any one of SEQ ID NO: 14, 16,
or 24, or any one of SEQ ID NO: 14, 18, or 24, or any one of SEQ ID
NO: 14, 20 or 24, or any one of SEQ ID NO: 14, 22, or 24, or any
one of SEQ ID NO: 16, 18, or 24, or any one of SEQ ID NO: 16, 20 or
24, or any one of SEQ ID NO: 16, 22, or 24, or any one of SEQ ID
NO: 18, 20 or 24, or any one of SEQ ID NO 18, 22, or 24.
[0100] In a further embodiment the 5' signal sequence portion
encodes fragments of at least 10, 12, 15, 18 or 20 amino acids of
any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
or 26 or any one of SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20, 22,
24, or 26, or any one of SEQ ID NO: 2, 4, 10, 12, 14, 16, 18, 20,
22, or 24, or SEQ ID NO: 24, or any one of SEQ ID NO: 2, 4, or 24,
or any one of SEQ ID NO: 2, 10, or 24, or any one of SEQ ID NO: 2,
12, or 24, or any one of SEQ ID NO: 2, 14, or 24, or any one of SEQ
ID NO: 4, 10 or 24, or any one of SEQ ID NO: 4, 12, or 24, or any
one of SEQ ID NO: 4, 16, or 24, or any one of SEQ ID NO: 4, 18 or
24, or any one of SEQ ID NO: 4, 20 or 24, or any one of SEQ ID NO:
4, 22, or 24, or any one of SEQ ID NO: 10, 12, or 24, or any one of
SEQ ID NO: 10, 14, or 24, or any one of SEQ ID NO: 10, 16, or 24,
or any one of SEQ ID NO: 10, 18, or 24, or any one of SEQ ID NO:
10, 22 or 24, or any one of SEQ ID NO: 12, 14, or 24, or any one of
SEQ ID NO: 12, 16, or 24, or any one of SEQ ID NO: 12, 18, or 24,
or any one of SEQ ID NO: 12, 20, or 24, or any one of SEQ ID NO:
12, 22, or 24, or any one of SEQ ID NO: 14, 16, or 24, or any one
of SEQ ID NO: 14, 18, or 24, or any one of SEQ ID NO: 14, 20 or 24,
or any one of SEQ ID NO: 14, 22, or 24, or any one of SEQ ID NO:
16, 18, or 24, or any one of SEQ ID NO: 16, 20 or 24, or any one of
SEQ ID NO: 16, 22, or 24, or any one of SEQ ID NO: 18, 20 or 24, or
any one of SEQ ID NO 18, 22, or 24 which are capable of directing
transport of a protein to the bacterial periplasm.
[0101] In a further aspect of the invention there is provided a
polynucleotide comprising a 5' signal sequence portion and a 3'
toxin portion, wherein the 5' signal portion sequence encodes a
signal peptide having an amino acid sequence of; [0102] (a) SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID
NO: 2, 4, 10 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID NO: NO:
2, 4, 10, 12, 14, 16, 18, 20, 22, or 24; or SEQ ID NO: 24, or SEQ
ID NO: 2, 4, or 24; or SEQ ID NO: 2, 10, or 24; or SEQ ID NO: 2,
12, or 24; or SEQ ID NO: 2, 14, or 24; or SEQ ID NO: 4, 10 or 24;
or SEQ ID NO: 4, 12, or 24; or SEQ ID NO: 4, 16, or 24; or SEQ ID
NO: 4, 18 or 24; or SEQ ID NO: 4, 20 or 24; or SEQ ID NO: 4, 22, or
24; or SEQ ID NO: 10, 12, or 24; or SEQ ID NO: 10, 14, or 24; or
SEQ ID NO: 10, 16, or 24; or SEQ ID NO: 10, 18, or 24; or SEQ ID
NO: 10, 22 or 24; or SEQ ID NO: 12, 14, or 24; or SEQ ID NO: 12,
16, or 24; or SEQ ID NO: 12, 18, or 24; or SEQ ID NO: 12, 20, or
24; or SEQ ID NO: 12, 22, or 24; or SEQ ID NO: 14, 16, or 24; or
SEQ ID NO: 14, 18, or 24; or SEQ ID NO: 14, 20 or 24; or SEQ ID NO:
14, 22, or 24; or SEQ ID NO: 16, 18, or 24; or SEQ ID NO: 16, 20 or
24; or SEQ ID NO: 16, 22, or 24; or SEQ ID NO: 18, 20 or 24; or any
one of SEQ ID NO 18, 22, or 24. [0103] (b) (variants of) SEQ ID NO:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID NO: 2,
4, 10 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID NO: NO: 2, 4,
10, 12, 14, 16, 18, 20, 22, or 24; or SEQ ID NO: 24, or SEQ ID NO:
2, 4, or 24; or SEQ ID NO: 2, 10, or 24; or SEQ ID NO: 2, 12, or
24; or SEQ ID NO: 2, 14, or 24; or SEQ ID NO: 4, 10 or 24; or SEQ
ID NO: 4, 12, or 24; or SEQ ID NO: 4, 16, or 24; or SEQ ID NO: 4,
18 or 24; or SEQ ID NO: 4, 20 or 24; or SEQ ID NO: 4, 22, or 24; or
SEQ ID NO: 10, 12, or 24; or SEQ ID NO: 10, 14, or 24; or SEQ ID
NO: 10, 16, or 24; or SEQ ID NO: 10, 18, or 24; or SEQ ID NO: 10,
22 or 24; or SEQ ID NO: 12, 14, or 24; or SEQ ID NO: 12, 16, or 24;
or SEQ ID NO: 12, 18, or 24; or SEQ ID NO: 12, 20, or 24; or SEQ ID
NO: 12, 22, or 24; or SEQ ID NO: 14, 16, or 24; or SEQ ID NO: 14,
18, or 24; or SEQ ID NO: 14, 20 or 24; or SEQ ID NO: 14, 22, or 24;
or SEQ ID NO: 16, 18, or 24; or SEQ ID NO: 16, 20 or 24; or SEQ ID
NO: 16, 22, or 24; or SEQ ID NO: 18, 20 or 24; or any one of SEQ ID
NO 18, 22, or 24 varying from the corresponding sequences by 1, 2
or 3 point mutations, amino acid insertions or amino acid deletions
which are capable of directing an expressed protein to the
periplasm; or [0104] (c) fragments of at least 10 amino acids of
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or
SEQ ID NO: 2, 4, 10 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID
NO: NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, or 24; or SEQ ID NO: 24,
or SEQ ID NO: 2, 4, or 24; or SEQ ID NO: 2, 10, or 24; or SEQ ID
NO: 2, 12, or 24; or SEQ ID NO: 2, 14, or 24; or SEQ ID NO: 4, 10
or 24; or SEQ ID NO: 4, 12, or 24; or SEQ ID NO: 4, 16, or 24; or
SEQ ID NO: 4, 18 or 24; or SEQ ID NO: 4, 20 or 24; or SEQ ID NO: 4,
22, or 24; or SEQ ID NO: 10, 12, or 24; or SEQ ID NO: 10, 14, or
24; or SEQ ID NO: 10, 16, or 24; or SEQ ID NO: 10, 18, or 24; or
SEQ ID NO: 10, 22 or 24; or SEQ ID NO: 12, 14, or 24; or SEQ ID NO:
12, 16, or 24; or SEQ ID NO: 12, 18, or 24; or SEQ ID NO: 12, 20,
or 24; or SEQ ID NO: 12, 22, or 24; or SEQ ID NO: 14, 16, or 24; or
SEQ ID NO: 14, 18, or 24; or SEQ ID NO: 14, 20 or 24; or SEQ ID NO:
14, 22, or 24; or SEQ ID NO: 16, 18, or 24; or SEQ ID NO: 16, 20 or
24; or SEQ ID NO: 16, 22, or 24; or SEQ ID NO: 18, 20 or 24; or any
one of SEQ ID NO 18, 22, or 24 which are capable of directing an
expressed protein to the periplasm; [0105] and the 3' toxin portion
encodes a bacterial toxin or fragment or variant thereof.
[0106] There is further provided a polynucleotide comprising a 5'
signal sequence portion and a 3' toxin portion, wherein the 5'
signal portion sequence encodes a signal peptide having an amino
acid sequence of [0107] (d) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, or 26; [0108] (e) variants of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, varying from the
corresponding sequences by 1, 2 or 3 point mutations, amino acid
insertions or amino acid deletions, which are capable of directing
an expressed protein to the periplasm; or [0109] (f) fragments of
at least 10 amino acids of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, or 26 which are capable of directing an expressed
protein to the periplasm;
[0110] and the 3' toxin portion encodes a bacterial toxin or
fragment or variant thereof.
[0111] In one embodiment the 5' signal sequence portion of the
polynucleotide of the invention encodes at least one of SEQ ID NO:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In a further
embodiment the 5' signal sequence portion of the polynucleotide of
the invention encodes at least one of SEQ ID NO: 2, 4, 10, 12, 14,
16, 18, 20, 22, 24, or 26. In one embodiment the 5' signal sequence
portion of the polynucleotide of the invention encodes at least one
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26; or
SEQ ID NO: 2, 4, 10 12, 14, 16, 18, 20, 22, 24, or 26; or SEQ ID
NO: NO: 2, 4, 10, 12, 14, 16, 18, 20, 22, or 24; or SEQ ID NO: 24,
or SEQ ID NO: 2, 4, or 24; or SEQ ID NO: 2, 10, or 24; or SEQ ID
NO: 2, 12, or 24; or SEQ ID NO: 2, 14, or 24; or SEQ ID NO: 4, 10
or 24; or SEQ ID NO: 4, 12, or 24; or SEQ ID NO: 4, 16, or 24; or
SEQ ID NO: 4, 18 or 24; or SEQ ID NO: 4, 20 or 24; or SEQ ID NO: 4,
22, or 24; or SEQ ID NO: 10, 12, or 24; or SEQ ID NO: 10, 14, or
24; or SEQ ID NO: 10, 16, or 24; or SEQ ID NO: 10, 18, or 24; or
SEQ ID NO: 10, 22 or 24; or SEQ ID NO: 12, 14, or 24; or SEQ ID NO:
12, 16, or 24; or SEQ ID NO: 12, 18, or 24; or SEQ ID NO: 12, 20,
or 24; or SEQ ID NO: 12, 22, or 24; or SEQ ID NO: 14, 16, or 24; or
SEQ ID NO: 14, 18, or 24; or SEQ ID NO: 14, 20 or 24; or SEQ ID NO:
14, 22, or 24; or SEQ ID NO: 16, 18, or 24; or SEQ ID NO: 16, 20 or
24; or SEQ ID NO: 16, 22, or 24; or SEQ ID NO: 18, 20 or 24; or any
one of SEQ ID NO 18, 22, or 24. The nucleotide sequences encoded by
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 may be
identical to the corresponding polynucleotide sequences of SEQ ID
NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, or 25. Alternatively
it may be any sequence, which as a result of the redundancy
(degeneracy) of the genetic code, also encodes polypeptides of SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26.
Alternatively the polynucleotide may comprise a portion that
encodes SEQ ID NO:33-45.
[0112] The polynucleotides of the invention may also comprise a 5'
signal sequence portion which encodes a variant of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 that is capable of
directing a protein to the periplasm.
[0113] The present invention also provides for a 5' signal sequence
portion comprising a fragment of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, or 26. Fragments of the invention consist of
contiguous portions of at least, 10, 15, or 20 amino acids from SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In an
embodiment the fragments lead to at least 50, 60, 70, 90, 90 or
100% of the polypeptide encoded toxin being transported to the
periplasm. In a further embodiment the fragments have the same or
substantially the same periplasmic transport properties as the
polypeptide comprising the corresponding amino acid sequence from
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26. In a
further embodiment the fragments are capable of directing
co-translational transport of a polypeptide to the periplasm.
[0114] The 3' toxin portion encodes any bacterial toxin. In one
embodiment the encoded bacterial toxin is diphtheria, tetanus,
pertussis or pneumolysin toxin.
[0115] In one embodiment the 3' toxin portion encodes a bacterial
toxoid for example Diphtheria toxoid or CRM197. Optionally the 3'
toxin portion encodes the amino acid sequence described in SEQ ID
NO:32. In a further embodiment the 3' toxin portion comprises the
nucleotide sequence of SEQ ID NO: 31. In an embodiment the
polypeptide encodes SEQ ID NO:33-45.
[0116] Alternatively the 3' portion may encode any fragment of at
least 10, 15, 25, 35, 50, 100, 150 amino acids or variant with 80%,
85%, 90%, 95%, 98% or 99% sequence homology of the above described
toxin or toxoid.
[0117] Optionally the 5' signal sequence portion is directly 5' of
the 3' toxin portion. Alternatively they are separated by at least
3, 6, 9, 12, 21, 51, 99, 300 or 1000 further nucleotides. For
example these nucleotides may encode one or more further peptide
sequences of at least 10, 20, 30, 50, 100, 200 or 500 amino
acids.
[0118] In addition for each and every polynucleotide of the
invention there is provided a polynucleotide complementary to it.
It is preferred that these complementary polynucleotides are fully
complementary to each polynucleotide with which they are
complementary.
[0119] Also the invention contemplates the expression of any of the
polynucleotides within a sequence coding for a larger protein such
as a precursor or a fusion protein. It is often advantageous to
include an additional amino acid sequence which contains secretory
or leader sequences, pro-sequences, sequences which aid in
purification such as multiple histidine residues, or an additional
sequence for stability during recombinant production. Furthermore,
addition of exogenous polypeptide or lipid tail or polynucleotide
sequences to increase the immunogenic potential of the final
molecule is also considered.
[0120] In an embodiment the 5' signal sequence portion is not the
tox signal sequence of C. diphtheriae.
[0121] Polypeptide of the Invention
[0122] The present invention also provides for polypeptides encoded
by the polynucleotides of the invention. In addition fragments and
variants of these polypeptides are provided.
[0123] The invention encompasses polypeptides encoding
polynucleotides of the invention.
[0124] Fragments of these polypeptides are also encompassed by the
invention. The fragments contain segments from both the 5' signal
sequence portion and 3' toxin portion. In a further embodiment
these fragments comprise at least 10, 15 or 20 amino acids of a
signal peptide. In an embodiment these fragments comprise at least
10, 15 or 20 of the amino acids from SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, or 26 or SEQ ID NO: 33-45.
[0125] In an embodiment these fragments are at least 50%, 60%, 70%,
80%, 90% or 100% directed to the periplasm when expressed in a gram
negative bacterium such as E. coli.
[0126] The invention also encompasses fusion proteins including the
polypeptide or polypeptide fragments of the invention.
[0127] The polypeptides, or immunogenic fragments, of the invention
may be a part of a larger protein such as a precursor or a fusion
protein. It is often advantageous to include an additional amino
acid sequence which contains sequences which aid in purification
such as multiple histidine residues, or an additional sequence for
stability during recombinant production. Furthermore, addition of
exogenous polypeptide or lipid tail or polynucleotide sequences to
increase the immunogenic potential of the final molecule is also
considered.
[0128] Vectors and Host Cells
[0129] The invention also relates to vectors that comprise a
polynucleotide or polynucleotides of the invention, host cells that
are genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the invention.
[0130] Recombinant polypeptides of the present invention may be
prepared by processes well known to those skilled in the art from
genetically engineered host cells comprising expression systems.
Accordingly, in a further aspect, the present invention relates to
expression systems that comprise a polynucleotide or
polynucleotides of the present invention, to host cells which are
genetically engineered with such expression systems, and to the
production of polypeptides of the invention by recombinant
techniques. In a further aspect of the invention the present
invention relates to a polynucleotide of the invention linked to an
inducible promoter such that when the promoter is induced a
polypeptide encoded by the polynucleotide is expressed. A further
aspect of the invention comprises said vector wherein the inducible
promoter is activated by addition of a sufficient quantity of IPTG.
Optionally this is at a concentration of between 0.1 and 10 mM, 0.1
and 5 mM, 0.1 and 2.5 mM, 0.2 and 10 mM, 0.2 and 5 mM, 0.2 and 2.5
mM, 0.4 and 10 mM, 1 and 10 mM, 1 and 5 mM, 2.5 and 10 mM, 2.5 and
5 mM, 5 and 10 mM.
[0131] For recombinant production of the polypeptides of the
invention, host cells can be genetically engineered to incorporate
expression systems or portions thereof or polynucleotides of the
invention. Introduction of a polynucleotide into the host cell can
be effected by methods described in many standard laboratory
manuals, such as Davis, et al., BASIC METHODS IN MOLECULAR BIOLOGY,
(1986) and Sambrook, et al., MOLECULAR CLONING: A LABORATORY
MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989), such as, calcium phosphate transfection,
DEAE-dextran mediated transfection, transvection, microinjection,
cationic lipid-mediated transfection, electroporation, conjugation,
transduction, scrape loading, ballistic introduction and
infection.
[0132] Representative examples of appropriate hosts include gram
negative bacterial cells, such as cells of, E. coli, Acinetobacter,
Actinobacillus, Bordetella, Brucella, Campylobacter, Cyanobacteria,
Enterobacter, Erwinia, Franciscella, Helicobacter, hemophilus,
Klebsiella, Legionella, Moraxella, Neisseria, Pasteurella, Proteus,
Pseudomonas, Salmonella, Serratia, Shigella, Treponema, Vibrio,
Yersinia. In a further aspect of the invention the host cell is an
Escherichia coli cell.
[0133] A great variety of expression systems can be used to produce
the polypeptides of the invention. In one embodiment the vector is
derived from bacterial plasmids. Generally, any system or vector
suitable to maintain, propagate or express polynucleotides and/or
to express a polypeptide in a host may be used for expression in
this regard. The appropriate DNA sequence may be inserted into the
expression system by any of a variety of well-known and routine
techniques, such as, for example, those set forth in Sambrook et
al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).
[0134] Fermentation
[0135] In a further aspect of the invention there is provided a
process for making a bacterial toxin comprising the steps of a)
growing a culture of the bacterial host cell of the invention and
b) inducing expression of the polypeptide such that a bacterial
toxoid is expressed periplasmically.
[0136] In a further embodiment of the invention there is provided a
process for periplasmic expression of a recombinant polypeptide by
[0137] A. Growing a culture of a gram-negative host cell; and
[0138] B. Inducing expression of a polypeptide such that a protein
is expressed periplasmically;
[0139] wherein one or more of the following steps is actioned
during expression: [0140] i. The pH of step a) is lower than the pH
of step b); [0141] ii. The temperature of step a) is higher than
the temperature of step b); or [0142] iii. The substrate feed rate
of step a) is higher than the substrate feed rate of step b).
[0143] In one embodiment step step i, step ii, step iii, step i and
step ii, step i and step iii, step ii and step iii or step i, step
ii and step iii are actioned. In a further embodiment a
polynucleotide of the invention is expressed.
[0144] Polypeptide expression is induced when an inducing agent
such as IPTG is added to a culture of host cells, causing
expression of polypeptide at an increased rate.
[0145] In one embodiment the pH of step a) is the same as the pH of
step b), in a second embodiment the pH of step a) is lower than the
pH of step b) i.e. the pH of step b) is made higher than that of
step a).
[0146] The person skilled in the art will recognise how to effect a
change between step a) and step b). A change in a condition (e.g.
pH, temperature or substrate feed rate) between step a) and step b)
means the general average condition during step a) or step b) is as
reported and may be assessed for instance, if there has been no
other intervention, just prior (e.g. 5 seconds, 15 seconds, 30
seconds, 1 minute, 15 minutes, 30 minutes or one hour) to induction
(step a) or just after (e.g. 5 seconds, 15 seconds, 30 seconds, 1
minute, 15 minutes, 30 minutes or one hour) induction (step b).
Clearly the inventors also envisage that the intervention to change
fermentation conditions may occur slightly before or slightly after
induction to achieve the same technical result but in such a
scenario again the general or average condition during step a) or
step b) will have changed as disclosed herein.
[0147] In a further embodiment the pH of step a) ranges from
5.0-7.0, 5.0-6.0, 6.0-7.0 or from 6.5-7.0.
[0148] In an embodiment the pH in step b) is maintained. In an
embodiment the pH is maintained at greater than pH 6.5, 7.0, 7.5,
8.0, 8.5, 9.0, 9.5, 10.0 or between 6.5 and 10.0, 6.5 and 9.5, 6.5
and 9.0, 6.5 and 8.5, 6.5 and 7.5, 6.5 and 7.0, 7.0 and 10.0, 7.0
and 9.5, 7.0 and 9.0, 7.0 and 8.5, 7.0 and 8.0, 7.0 and 7.5, 7.5
and 10.0, 7.5 and 9.5, 7.5 and 9.0, 7.5 and 8.5, 7.5 and 8.0, 8.0
and 10.0, 8.0 and 9.5, 8.0 and 9.0, 8.0 and 8.5, 8.5 and 10.0, 8.5
and 9.5, 8.0 and 9.0, 8.0 and 8.5, 8.5 and 10.0, 8.5 and 9.5, 8.5
and 9.0, 9.0 and 10.0, 9.0 and 9.5 or 9.5 and 10.0. In a further
embodiment the pH is maintained using a buffer from the group
consisting of phosphate buffer, Tris buffer and histidine buffer.
Optionally the buffer is at a concentration of 10-200 mM, 50-100
mM, 100-200 mM, 10-50 mM or 50-150 mM. Optionally the buffer is
phosphate buffer at 80-120 mM, 80-100 mM or 100 mM.
[0149] In one embodiment the pH in step b) is at least, exactly or
approximately 2.0, 1.5, 1.0, 0.5, 0.3, 0.2 or 0.1 pH units higher
than the pH in step a).
[0150] In one embodiment the process of the invention is carried
out in a fermentor. In an embodiment the pH is increased such that
the pH in step b) is higher than the pH of step a). Optionally this
increase in pH is achieved by addition of base for instance sodium
hydroxide or ammonia.
[0151] In a further embodiment the pH of step (b) is maintained
between 6.5 and 10.0, 6.5 and 9.5, 6.5 and 9.0, 6.5 and 8.5, 6.5
and 7.5, 6.5 and 7.0, 7.0 and 10.0, 7.0 and 9.5, 7.0 and 9.0, 7.0
and 8.5, 7.0 and 8.0, 7.0 and 7.5, 7.5 and 10.0, 7.5 and 9.5, 7.5
and 9.0, 7.5 and 8.5, 7.5 and 8.0, 8.0 and 10.0, 8.0 and 9.5, 8.0
and 9.0, 8.0 and 8.5, 8.5 and 10.0, 8.5 and 9.5, 8.0 and 9.0, 8.0
and 8.5, 8.5 and 10.0, 8.5 and 9.5, 8.5 and 9.0, 9.0 and 10.0, 9.0
and 9.5 or 9.5 and 10.0 by the addition of base for instance sodium
hydroxide or ammonia during step b).
[0152] In one embodiment the process of the invention comprises a
first substrate feed rate in step a) and a second substrate feed
rate in step b) wherein the second substrate feed rate is lower
than the first substrate feed rate. In a further embodiment the
second substrate feed rate is maintained between 5% and 90%, 20%
and 80% or 20% and 30% of the substrate feed rate maintained during
step a).
[0153] Substrate feed rate (or substrate provision rate) is the
rate of substrate addition during the fed-batch and induction
phases (ml min.sup.-10) i.e. does not include any initial non-fed
batch phase period of fermentation.
[0154] In an embodiment the bacterial toxin is pneumolysin toxin,
diphtheria toxin, a tetanus toxin or a pertussis toxin or a point
mutated variant thereof. In an embodiment the toxin is diptheria
toxoid. In a further embodiment the toxin is CRM197, optionally
this encoded by SEQ ID NO:32. In a further embodiment the bacterial
toxin is a fragment or point mutated variant of pneumolysin,
diphtheria toxin, tetanus toxin or pertussis toxin.
[0155] The bacterial host cell of this process was defined in the
section on vectors and host cells. In one embodiment the bacterial
host cell is selected from the group consisting of E. coli,
Acinetobacter, Actinobacillus, Bordetella, Brucella, Campylobacter,
Cyanobacteria, Enterobacter, Erwinia, Franciscella, Helicobacter,
Hemophilus, Klebsiella, Legionella, Moraxella, Neisseria,
Pasteurella, Proteus, Pseudomonas, Salmonella, Serratia, Shigella,
Treponema, Vibrio, and Yersinia.
[0156] In an embodiment the bacterial host cell is a strain of E.
coli. In a further embodiment the bacterial host cell is a K strain
or a B strain of E. coli. In a further embodiment the bacterial
host cell is a K12 or B834 strain of E. coli.
[0157] In an embodiment the temperature of step a) is higher than
the temperature of step b). In an embodiment step a) of the process
of the invention is carried out at a temperature of 20-40.degree.
C. Optionally step b) of the process of the invention is carried
out at a temperature of 20-28.degree. C., 21-27.degree. C.,
22-26.degree. C., 23-24.degree. C., 21-24.degree. C., or
22-23.degree. C.
[0158] In a further embodiment expression is induced in step b) by
the addition of a sufficient quantity of IPTG
(isopropyl-beta-D-thiogalactopyranoside). Optionally this is at a
concentration of between 0.1 and 10 mM, 0.1 and 5 mM, 0.1 and 2.5
mM, 0.2 and 10 mM, 0.2 and 5 mM, 0.2 and 2.5 mM, 0.4 and 10 mM, 1
and 10 mM, 1 and 5 mM, 2.5 and 10 mM, 2.5 and 5 mM, 5 and 10
mM.
[0159] In one embodiment the optical density OD.sub.650 of the
bacteria is between 0-2.5 or between 0.4-1.5 at induction. The term
`at induction` refers to the point in the process at which an
inducing agent such as IPTG is added, this will occur at the very
beginning of step b).
[0160] A fermentor is any apparatus suitable for the industrial
production of bacterial cultures. However this term does not
include culture flasks which are typically used for growth of
bacteria on a smaller scale. Optionally the fermentor contains
20-500, 50-500, 100-500, 250-500, 400-500, 20-400, 50-400, 100-400,
250-400, 20-250, 100-200, 100-250, 250-300, 300-500, 10-2000,
500-2000, 1000-2000, 1500-2000 or 1000-1500 or around 150 litres of
culture.
[0161] Optionally the culture in the fermentor is agitated.
Agitation is optionally by stirring the culture in the fermentor
but may be by any other suitable means, for example by agitation,
vibromixer and/or gas bubbling.
[0162] In an embodiment the dissolved oxygen level (DO) in the
fermentor is (maintained) greater than 5%, 10%, 15%, 16%, 17%, 18%,
19% 20%, 21%, 22%, 23%, 24%, 25% or 30%. In a further embodiment
the DOin the fermentor is 5%-50%, 10%-40%, 15%-25% or 17%-22%. A
100% DO is the amount of oxygen present when the medium (in the
absence of a culture) is saturated with oxygen following bubbling
compressed air through the medium at 28.degree. C. and pressure of
0.5 bar.
[0163] The fermentation step may be subject to a large amount of
foam production. In order to control foam formation an antifoam
agent is optionally added to the fermentor. Optionally a foam probe
or mechanical foam breaker is used in the fermentor, this may be
used as well as the antifoam agent.
[0164] In an embodiment the process for making the bacterial toxin
involves removal of a signal peptide from the bacterial toxin
within the bacterial host cell to obtain a mature bacterial toxin.
Optionally this removal is carried out by host cell machinery. In
an embodiment cleavage is performed by the E. coli signal
peptidase. Optionally the removed signal sequence is cleaved
further by signal peptide peptidase.
[0165] In an embodiment the process of the invention comprises a
further step c) of harvesting the cell paste from the culture.
[0166] In an embodiment centrifugation is used to harvest the
cells. Optionally this takes place at 5,000-8,000 g, 5,500-7,500 g,
6,000-7,000 g. Optionally this takes place between 4.degree.
C.-10.degree. C. 5.degree. C.-9.degree. C., 6.degree. C.-8.degree.
C. or 7.degree. C.-8.degree. C.
[0167] In a further embodiment the process of the invention
comprises a further step of purifying the bacterial toxin, for
example as a mature bacterial toxin. In an embodiment this step
involves cell disruption and further purification using
chromatography and filtration techniques.
[0168] In an embodiment cells are disrupted using osmotic shock,
mechanical or enzymatic methods. Optionally the mechanical method
comprises using a mechanical homogenizer, vortexing, sonication,
using a French press or bead milling. Optionally the enzymatic
method comprises using lysozyme, zymolase or lysostaphin
digestion.
[0169] In an embodiment the chromatography technique is affinity
chromatography, gel filtration, high pressure liquid chromatography
(HPLC) or ion exchange chromatography. Optionally the affinity
chromatography uses an affinity tag purification column, an
antibody purification column, a lectin affinity column, a
prostaglandin purification column or a streptavidin column.
Optionally the HPLC uses an ion exchange column, a reverse phase
column or a size exclusion column. Optionally the ion exchange
column is an anion exchange column or a cation exchange column.
[0170] Conjugation
[0171] The invention also provides a process for making a conjugate
comprising the steps of i) making a bacterial toxin using the
process of the invention, and b) conjugating the bacterial toxin of
step ii) to an antigen.
[0172] In an embodiment the antigen is a bacterial saccharide. For
example the bacterial saccharide is a capsular saccharide from a
bacterium selected from the group consisting of S. pneumoniae, H.
influenzae, N. meningitidis, group B Streptococcus, group A
Streptococcus, Salmonella Vi, enterococci and S. aureus. As defined
herein a "saccharide" may be either an oligosaccharide or a
polysaccharide.
[0173] The saccharide conjugates present in the immunogenic
compositions of the invention may be prepared by any known coupling
technique. The conjugation method may rely on activation of the
saccharide with 1-cyano-4-dimethylamino pyridinium
tetrafluoroborate (CDAP) to form a cyanate ester. The activated
saccharide may thus be coupled directly or via a spacer (linker)
group to an amino group on the carrier protein. For example, the
spacer could be cystamine or cysteamine to give a thiolated
polysaccharide which could be coupled to the carrier via a
thioether linkage obtained after reaction with a
maleimide-activated carrier protein (for example using GMBS) or a
haloacetylated carrier protein (for example using iodoacetimide
[e.g. ethyl iodoacetimide HCl] or N-succinimidyl bromoacetate or
SIAB, or SIA, or SBAP). Preferably, the cyanate ester (optionally
made by CDAP chemistry) is coupled with hexane diamine or ADH and
the amino-derivatised saccharide is conjugated to the carrier
protein using carbodiimide (e.g. EDAC or EDC) chemistry via a
carboxyl group on the protein carrier. Such conjugates are
described in PCT published application WO 93/15760 Uniformed
Services University and WO 95/08348 and WO 96/29094.
[0174] Other suitable techniques use carbodiimides, hydrazides,
active esters, norborane, p-nitrobenzoic acid,
N-hydroxysuccinimide, S-NHS, EDC, TSTU. Many are described in WO
98/42721. Conjugation may involve a carbonyl linker which may be
formed by reaction of a free hydroxyl group of the saccharide with
CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4, Hearn et al J.
Chromatogr. 1981. 218; 509-18) followed by reaction with a protein
to form a carbamate linkage. This may involve reduction of the
anomeric terminus to a primary hydroxyl group, optional
protection/deprotection of the primary hydroxyl group' reaction of
the primary hydroxyl group with CDI to form a CDI carbamate
intermediate and coupling the CDI carbamate intermediate with an
amino group on a protein.
[0175] The conjugates can also be prepared by direct reductive
amination methods as described in U.S. Pat. No. 4,365,170
(Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods
are described in EP-0-161-188, EP-208375 and EP-0-477508.
[0176] A further method involves the coupling of a cyanogen bromide
(or CDAP) activated saccharide derivatised with adipic acid
dihydrazide (ADH) to the protein carrier by Carbodiimide
condensation (Chu C. et al Infect. Immunity, 1983 245 256), for
example using EDAC.
[0177] In an embodiment, a hydroxyl group (preferably an activated
hydroxyl group for example a hydroxyl group activated to make a
cyanate ester [e.g. with CDAP]) on a saccharide is linked to an
amino or carboxylic group on a protein either directly or
indirectly (through a linker). Where a linker is present, a
hydroxyl group on a saccharide is preferably linked to an amino
group on a linker, for example by using CDAP conjugation. A further
amino group in the linker for example ADH) may be conjugated to a
carboxylic acid group on a protein, for example by using
carbodiimide chemistry, for example by using EDAC. In an
embodiment, the pneumococcal capsular saccharide(s) is conjugated
to the linker first before the linker is conjugated to the carrier
protein. Alternatively the linker may be conjugated to the carrier
before conjugation to the saccharide.
[0178] In general the following types of chemical groups on a
protein carrier can be used for coupling/conjugation:
[0179] A) Carboxyl (for instance via aspartic acid or glutamic
acid). In one embodiment this group is linked to amino groups on
saccharides directly or to an amino group on a linker with
carbodiimide chemistry e.g. with EDAC.
[0180] B) Amino group (for instance via lysine). In one embodiment
this group is linked to carboxyl groups on saccharides directly or
to a carboxyl group on a linker with carbodiimide chemistry e.g.
with EDAC. In another embodiment this group is linked to hydroxyl
groups activated with CDAP or CNBr on saccharides directly or to
such groups on a linker; to saccharides or linkers having an
aldehyde group; to saccharides or linkers having a succinimide
ester group.
[0181] C) Sulphydryl (for instance via cysteine). In one embodiment
this group is linked to a bromo or chloro acetylated saccharide or
linker with maleimide chemistry. In one embodiment this group is
activated/modified with bis diazobenzidine.
[0182] D) Hydroxyl group (for instance via tyrosine). In one
embodiment this group is activated/modified with bis
diazobenzidine.
[0183] E) Imidazolyl group (for instance via histidine). In one
embodiment this group is activated/modified with bis
diazobenzidine.
[0184] F) Guanidyl group (for instance via arginine).
[0185] G) Indolyl group (for instance via tryptophan).
[0186] On a saccharide, in general the following groups can be used
for a coupling: OH, COOH or NH2. Aldehyde groups can be generated
after different treatments known in the art such as: periodate,
acid hydrolysis, hydrogen peroxide, etc.
[0187] Direct Coupling Approaches:
[0188] Saccharide-OH+CNBr or CDAP----->cyanate
ester+NH2-Prot---->conjugate
[0189] Saccharide-aldehyde+NH2-Prot---->Schiff
base+NaCNBH3---->conjugate
[0190] Saccharide-COOH+NH2-Prot+EDAC---->conjugate
[0191] Saccharide-NH2+COOH-Prot+EDAC---->conjugate
[0192] Indirect Coupling Via Spacer (Linker) Approaches:
[0193] Saccharide-OH+CNBr or CDAP--->cyanate
ester+NH2----NH2---->saccharide----NH2+COOH-Prot+EDAC----->conjugat-
e
[0194] Saccharide-OH+CNBr or CDAP---->cyanate
ester+NH2-----SH----->saccharide----SH+SH-Prot (native Protein
with an exposed cysteine or obtained after modification of amino
groups of the protein by SPDP for
instance)----->saccharide-S-S-Prot
[0195] Saccharide-OH+CNBr or CDAP--->cyanate
ester+NH2----SH------->saccharide----SH+maleimide-Prot
(modification of amino groups)---->conjugate
[0196] Saccharide-OH+CNBr or CDAP--->cyanate
ester+NH2-----SH--->Saccharide-SH+haloacetylated-Prot---->Conjugate
[0197]
Saccharide-COOH+EDAC+NH2-----NH2--->saccharide------NH2+EDAC+COO-
H-Prot---->conjugate
[0198]
Saccharide-COOH+EDAC+NH2----SH----->saccharide----SH+SH-Prot
(native Protein with an exposed cysteine or obtained after
modification of amino groups of the protein by SPDP for
instance)----->saccharide-S-S-Prot
[0199]
Saccharide-COOH+EDAC+NH2----SH----->saccharide----SH+maleimide-P-
rot (modification of amino groups)---->conjugate
[0200]
Saccharide-COOH+EDAC+NH2----SH--->Saccharide-SH+haloacetylated-P-
rot---->Conjugate
[0201]
Saccharide-Aldehyde+NH2-----NH2---->saccharide---NH2+EDAC+COOH-P-
rot---->conjugate
[0202] Note: instead of EDAC above, any suitable carbodiimide may
be used.
[0203] In summary, the types of protein carrier chemical group that
may be generally used for coupling with a saccharide are amino
groups (for instance on lysine residues), COOH groups (for instance
on aspartic and glutamic acid residues) and SH groups (if
accessible) (for instance on cysteine residues.
[0204] Vaccine or Immunogenic Compositions of the Invention
[0205] The present invention further provides a process for
manufacturing a vaccine or immunogenic composition comprising the
steps of [0206] A. making a bacterial toxin or conjugate using the
process of the invention and; [0207] B. mixing the bacterial toxin
or conjugate thereof with a pharmaceutically acceptable
excipient.
[0208] The vaccine or immunogenic composition produced by this
process may additionally comprise antigens from further bacterial
species. In one embodiment the vaccine or immunogenic composition
may comprise antigens selected from S. pneumoniae, H. influenzae,
N. meningitidis, E. coli, M. cattarhalis, tetanus, diphtheria,
pertussis, S. epidermidis, enterococci, or S. aureus.
[0209] In a further step the polypeptides of the invention may be
mixed with an adjuvant. The choice of a suitable adjuvant to be
mixed with bacterial toxins or conjugates made using the processes
of the invention is within the knowledge of the person skilled in
the art. Suitable adjuvants include an aluminium salt such as
aluminium hydroxide gel or aluminum phosphate or alum, but may also
be other metal salts such as those of calcium, magnesium, iron or
zinc, or may be an insoluble suspension of acylated tyrosine, or
acylated sugars, cationically or anionically derivatized
saccharides, or polyphosphazenes.
[0210] The vaccine preparations containing immunogenic compositions
of the present invention may be used to protect or treat a mammal
susceptible to infection, by means of administering said vaccine
via systemic or mucosal route. These administrations may include
injection via the intramuscular, intraperitoneal, intradermal or
subcutaneous routes; or via mucosal administration to the
oral/alimentary, respiratory, genitourinary tracts. Intranasal
administration of vaccines for the treatment of pneumonia or otitis
media is preferred (as nasopharyngeal carriage of pneumococci can
be more effectively prevented, thus attenuating infection at its
earliest stage). Although the vaccine of the invention may be
administered as a single dose, components thereof may also be
co-administered together at the same time or at different times
(for instance pneumococcal saccharide conjugates could be
administered separately, at the same time or 1-2 weeks after the
administration of the any bacterial protein component of the
vaccine for optimal coordination of the immune responses with
respect to each other). In addition to a single route of
administration, 2 different routes of administration may be used.
For example, saccharides or saccharide conjugates may be
administered IM (or ID) and bacterial proteins may be administered
IN (or ID). In addition, the vaccines of the invention may be
administered IM for priming doses and IN for booster doses.
[0211] The content of toxins in the vaccine will typically be in
the range 1-100 .mu.g, preferably 5-50 .mu.g, most typically in the
range 5-25 .mu.g. Following an initial vaccination, subjects may
receive one or several booster immunizations adequately spaced.
[0212] Vaccine preparation is generally described in Vaccine Design
("The subunit and adjuvant approach" (eds Powell M. F. & Newman
M. J.) (1995) Plenum Press New York). Encapsulation within
liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
EXAMPLES
Example 1 Design of CRM197-Signal Sequence Constructs
[0213] Plasmids containing a sequence encoding an N-terminal signal
sequence fused to the CRM197 toxoid were created using standard
molecular biology techniques. Thirteen different signal sequences
were used (table 2); these were selected from the GenEMBL
database.
TABLE-US-00002 TABLE 2 Signal SEQ ID NO SEQ ID NO sequence
nucleotide Amino acid Origin PhtE 1 2 S. pneumoniae SipA 3 4 Group
B Strep OmpA 5 6 E. coli NspA 7 8 N. meningitidis TorT 9 10 E. coli
SfmC 11 12 E. coli FocC 13 14 E. coli Ccmh 15 16 E. coli Yra1 17 18
E. coli TolB 19 20 E. coli NikA 21 22 E. coli FlgI 23 24 E. coli
DsbA 25 26 E. coli
[0214] The first construct was created from three plasmids (see
FIG. 1). Plasmid A, created by Geneart, contained the DsbA signal
sequence (SEQ ID NO:25) 3' to a sequence encoding an N-terminal
fragment of CRM197 (amino acids 1-206 of SEQ ID NO 32). This
plasmid was cloned into the pET26b backbone plasmid (Novagen) by
digestion of both plasmids using NdeI and SacI restrictions enzymes
followed by ligation. Plasmid B, created by Geneart, contained a
C-terminal fragment of CRM197 (amino acids 206-533 of SEQ ID NO
32). this was cloned into the pET26b backbone using restriction
with AatII and XhoI restriction enzymes, the resulting plasmid was
named pRIT16668. This construct contains the signal sequence from
DsbA up to the signalase binding site, followed by two amino acids
from DsbA found after the signalase binding sequence, a further two
amino acids from the restriction enzyme binding site, and finally
CRM197 missing the first two amino acids. This means that when
these constructs are expressed the protein produced after
periplasmic expression and removal of the signal sequence by
signalase contains CRM197 missing the first two amino acids but
with an extra four amino acids from DsbA and the restriction
site.
[0215] The pRIT16668 plasmid contains NdeI and AgeI restrictions
sites flanking the DsbA signal sequence.
[0216] Plasmids containing the 12 other signal sequences were
created by digesting plasmid pRIT16668 with NdeI and AgeI to remove
the DsbA signal sequence. Hybridized oligonucleotides encoding each
of the other 12 signal sequences were, one at a time, ligated
between these two sites.
[0217] The resulting plasmids were transformed into Novablue
chemically competent cells (Novagen cat 70181-3, used as
recommended by manufacturer). The presence of the correct insert
was confirmed for each plasmid by sequencing. The plasmids were
then transformed individually into B834(DE3) chemically competent
cells (Novagen cat 69041-3HMS174 (DE3) chemically competent cells
or alternatively BLR(DE3) chemically competent cells (Novagen cat
69053-3, used as recommended by manufacturer), for expression.
Example 2 Design of a CRM197 Construct for Cytoplasmic Expression
(Lacking the Periplasmic Signal Sequence)
[0218] In this experiment, a plasmid similar to those created in
Example 1 but lacking the periplasmic signal sequence was created.
This design process is summarised in FIG. 2. Firstly the CRM197
sequence (SEQ ID NO:31) was amplified by standard PCR techniques
using two primers which were named MDSCRM1 (SEQ ID NO:27) and
MDSCRM2 (SEQ ID NO:28) and pRIT16668 as the template (the
temperature cycles used were (94.degree. C. 2'-55.degree. C.
2'-72.degree. C. 2'30).times.25-72.degree. C. 10'-end 4.degree.
C.).
[0219] This fragment was inserted into plasmid pRIT16668 in place
of the CRM197 signal sequence insert. This was achieved using
standard molecular biology techniques through digestion of the PCR
product and pRIT16668 with the restriction enzymes NdeI and XhoI.
The resulting plasmid contains the mature CRM197 sequence (SEQ ID
NO:31) but contains no signal sequence and was named pRIT16669.
[0220] The resulting plasmids were transformed into Novablue
chemically competent cells (Novagen cat 70181-3, used as
recommended by manufacturer). The presence of the correct insert
was confirmed for each plasmid by sequencing. The plasmids were
then transformed into B834(DE3) chemically competent cells (Novagen
cat 69041-3, used as recommended by manufacturer) for
expression.
Example 3 Design of an Optimal Signal Sequence-CRM197 Construct
[0221] The signal sequence FlgI was selected for use to create an
improved construct. The cloning process followed is summarized in
FIG. 3.
[0222] A region of DNA containing the FlgI signal sequence fused to
the N-terminal part of the CRM197 sequence was amplified using
standard PCR techniques. Two primers were used, primers named FlgI
c-CRMopt3e (SEQ ID NO:29) and MDSCRM906NC (SEQ ID NO: 30) primer
using pRIT16669 as template with cycling (94.degree. C.
2'(94.degree. C. 1'-60.degree. C. 1'-72.degree. C. 2'30).times.3
(94.degree. C. 1'-58.degree. C. 1'-72.degree. C. 2'30).times.3
(94.degree. C. 1'-56.degree. C. 1'-72.degree. C.
2'30).times.3(94.degree. C. 1'-54.degree. C. 1'-72.degree. C.
2'30).times.20 72.degree. C. 10'-end 4.degree. C.).
[0223] This fragment was inserted into plasmid pRIT16669 using
standard molecular biology techniques through digestion of the PCR
product and plasmid pRIT16669 with the restriction enzymes NdeI and
AatII. The resulting plasmid contains the complete mature
N-terminus of CRM197 (SEQ ID NO:31) and the FlgI signal sequence
terminating at the signalase binding site (SEQ ID NO:23) and was
named pRIT16681.
[0224] The resulting plasmids were transformed into Novablue
chemically competent cells (Novagen cat 70181-3, used as
recommended by manufacturer). The presence of the correct insert
was confirmed for each plasmid by sequencing. The plasmids were
then transformed into B834(DE3) chemically competent cells (Novagen
cat 69041-3) cells for expression.
Example 4 Expression of DsbA Signal Sequence-CRM197 Construct
[0225] In this experiment, cells transformed with the DsbA signal
sequence constructs created in Example 1 were cultured and
expression induced. Pre-cultures (3 ml) of the B834(DE3),
HMS174(DE3) and BLR(DE3) recombinant strain were grown in LBT
medium supplemented with 1% glucose in the presence of kanamycin
(50 .mu.g/ml) overnight at 37.degree. C. under agitation. The next
day samples of this preculture were added to 20 ml LBT medium with
50 .mu.g/ml of kanamycin after the optical density OD.sub.620 of
the medium reached 0.1. These cells were allowed to grow at
30.degree. C. under agitation. In this plasmid, CRM197 expression
is under the control of the lac operator, therefore expression of
the encoded CRM197 can be induced on addition of isopropyl-beta-D
thiogalactopyranoside (IPTG). When the optical density reached 0.6
induction with IPTG (added until there is a final concentration of
1 mM) was carried out and the culture was allowed to grow further,
for 3 h at 30.degree. C.
[0226] The level of CRM197 expression was evaluated by running the
total bacterial cell product on an SDS-PAGE gel (Novex Tris-glycine
10-20%) and staining with Coomassie brilliant blue. A Western blot
was carried out using mouse anti-DTPa polyclonal antibody staining
with NBT-BCIP.
[0227] FIG. 4 describes the results of this experiment. The
Coomassie gel shows no difference in expression profile between the
induced and non-induced cultures. The Western blot demonstrates a
lack of CRM197 in both the induced and non-induced cultures. There
is no or little periplasmic CRM197 expressed under these
conditions.
Example 5 Optimisation of pH and Temperature Conditions and
Expression Analysis of the Constructs
[0228] The steps in example 4 were repeated; however in this case
100 mM of K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer was added to the
culture before induction such that the medium was buffered to
pH7.5. Expression was induced by addition of 1 mM IPTG and the
culture was allowed to grow overnight at either 23.degree. C. or
30.degree. C. The level of protein expressed in the supernatant and
pellet was evaluated using Coomassie staining and Western blot
techniques as described in Example 4.
[0229] With regard to the post-translational signal sequences, the
expression level of CRM197 was very high for the OmpA signal
sequence, however the protein is cleaved to form a protein of
around 27 kDa. This truncated form is cell associated (not found in
either the periplasmic release fraction or in the culture medium).
With regard to the NspA signal sequence, expression is positive but
a low level of mature CRM197 is obtained.
[0230] With regard to the co-translational sequences, the
expression level is high with FlgI and SipA signal sequences,
however the CRM197 produced is not fully matured. Expression with
the NikA signal sequence leads to expression of mature protein. The
global expression level is lower than with FlgI and SipA signal
sequence but is still detectable in the total extract by Coomassie
staining.
Example 6 Expression Analysis of the CRM197 Cytoplasmic Expression
Constructs
[0231] DE3 cells were transformed with the plasmids produced in
example 2, cultured and expression of CRM197 induced as described
in Example 4. The CRM197 expression level was evaluated by running
the product of the total bacterial cell on an SDS-PAGE gel and
using Coomassie blue staining and western blot techniques as
described in Example 4. Bacterial extracts were performed by one
shot technic (Constant system) in standard PBS buffer. After
centrifugation, the soluble and insoluble fractions obtained were
loaded onto a gel.
[0232] The results of this experiment are presented in FIG. 6. The
gene is well expressed in these strains nevertheless a substantial
part of the protein is insoluble and remains in pellet. Furthermore
cytoplasmic expression leads to apparent lower molecular weight
proteins appearing on the gel.
Example 7 Expression Analysis of the Optimised FlgI Construct
[0233] BLR(DE3) E. coli cells were transformed with the construct
produced in example 3, cultured and expression induced as described
in the optimised protocol of example 5. In addition some sample
cells were grown without the addition of 100 mM
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer. Similarly the level of
protein expressed was evaluated using Coomassie staining and
Western blot techniques as described in Example 4. At induction
temperature was maintained at either 23.degree. C. or 30.degree. C.
and samples were taken at the time of induction, 2 hours after
induction, 4 hours after induction and after overnight
induction.
[0234] The results of this experiment are shown in FIG. 7. The
Western blot demonstrates an easily detectable level of expression
of CRM197 with buffer at 23.degree. C. The levels of protein
expression in the cells which were not buffered during the
induction phase were observed to be lower.
Example 8--Escherichia coli B2355 Pre-Culture
[0235] A pre-culture was prepared using a frozen seed culture of
Escherichia coli strain B2355. This strain is a B834(DE3) strain
transformed with a pET26b derivative containing a sequence coding
for a fusion protein between the signal peptide of FlgI from E.
coli and the mature part of CRM197 (this is plasmid pRIT16681
described in FIG. 3 and example 3). The seed culturability was
determined as approximately 1.times.10.sup.10 colony forming units
per ml.
[0236] The seed culture was thawed to room temperature and 400
.mu.l were used to inoculate a 2 litre Erlenmeyer flask containing
400 ml of preculture medium (adapted from Zabriskie et al. (J. Ind.
Microbiol. 2:87-95 (1987))).
[0237] The inoculated flask was then incubated at 37.degree. C.
(.+-.1.degree. C.) and 200 rpm. The pre-culture was stopped when
the optical density at 650 nm (OD.sub.650 nm) reached 2.50, (around
6 h of incubation). The pre-culture was used to inoculate medium in
a fermenter as soon as the culture was stopped (example 9).
Example 9--20 L Scale Fedbatch Fermentation
[0238] Method
[0239] A 20 litre fermenter (Biolafitte) was used. Nine litres of
batch phase medium were aseptically transferred into the fermenter
(adapted from Zabriskie et al. (J. Ind. Microbiol. 2:87-95 (1987)).
The pH of the medium was readjusted to 6.8 with base addition. 3 ml
of undiluted irradiated antifoam (SAG 471) was also added to the
fermenter. The temperature (28.degree. C.), head pressure (0.5
bar), aeration rate (20 litres sparged air per minute) and initial
agitation speed (300 rpm) were then set prior to inoculation. The
level of dissolved oxygen in these conditions was 100%. The head
pressure and aeration rate were maintained at a constant level
during the fermentation.
[0240] Inoculation was achieved by the addition of 18 ml of
pre-culture (prepared as described in Example 8).
[0241] During batch phase (0-15 h), the temperature was maintained
at 28.degree. C. The level of dissolved oxygen was set at 20%. The
level of dissolved oxygen (DO) was regulated by increasing stirring
when the DO fell below 20%. Glucose exhaustion resulted in an
increase in DO and a concomitant decrease in stirring.
[0242] After 15 h fermentation, additional substrate was added
according to the following feed addition profile:
TABLE-US-00003 TABLE 3 Additional Fermentation substrate feed rate
Cumulative time (h) (ml/min) weight fed (g) 0 0.000 0 1 0.000 0 2
0.000 0 3 0.000 0 4 0.000 0 5 0.000 0 6 0.000 0 7 0.000 0 8 0.000 0
9 0.000 0 10 0.000 0 11 0.000 0 12 0.000 0 13 0.000 0 14 0.000 0 15
0.000 0 16 0.600 21 17 1.150 81 18 1.150 161 19 1.150 241 20 1.150
321 21 1.150 400 22 1.150 480 23 1.150 560 24 1.150 639 25 1.150
719 26 1.150 799 27 1.150 878 28 1.150 958 29 1.150 1038 30 1.150
1117 31 1.150 1197 32 1.150 1277 33 1.150 1357 34 1.150 1436 35
1.150 1516 36 1.150 1596 37 1.150 1675 38 1.150 1755 39 1.150 1835
40 1.150 1914 41 1.150 1994 42 1.150 2074 43 1.150 2153 44 1.150
2233 45 1.150 2313 46 1.150 2393 47 0.325 2444 48 0.325 2466 49
0.325 2489 50 0.325 2511 51 0.325 2534 52 0.325 2556 53 0.325 2579
54 0.325 2601 55 0.325 2624 56 0.325 2646 57 0.325 2669 58 0.325
2691 59 0.325 2714 60 0.325 2736 61 0.325 2759 62 0.325 2782 63
0.325 2804 64 0.325 2827 65 0.325 2849 66 0.325 2872 67 0.325 2894
68 0.325 2917 69 0.325 2939 70 0.325 2962 71 0.325 2984 72 0.325
3007
[0243] During the fed-batch phase (15-46 h), the pH was maintained
at 6.8 by addition of base, the temperature was regulated at
28.degree. C., and the DO level was maintained at 20% through
control of the stirring rate.
[0244] At 46 hours IPTG was added to a final concentration of 1 mM
to induce the bacteria. In addition the pH is gradually increased
after 46 hours by addition of base, and the temperature was
decreased to 23.degree. C. (these changes are required for high
levels of periplasmic expression). The pH and temperature were
maintained during the whole induction phase (46-72 h). The DO level
was maintained at 20% by controlling the stirring rate.
[0245] At the end of the induction phase (72 h), cell paste was
collected by centrifugation (6,500.times.g, 4.degree. C. for 1 h),
and stored at -20.degree. C.
[0246] Periplasmic extraction was performed by osmotic shock using
a procedure adapted from Chen et al. (Biochem. Eng. J. 19:211-215
(2004)). CRM197 content in the periplasmic and cytoplasmic
fractions were assayed by Elisa.
[0247] FIG. 8 shows a typical fermentation profile with the process
parameters monitored during 20 L-scale fed-batch fermentation.
[0248] At the end of fermentation, periplasmic CRM197 productivity
was assayed by Elisa:
TABLE-US-00004 TABLE 4 Periplasmic Cytoplasmic Secretion efficiency
3180 mg/L 394 mg/L 87%
[0249] This technique demonstrated unprecedented levels of
expression and efficiency of secretion.
Example 10--Determination of the Optimum Feed Rates and
Temperatures to be Used During the Induction Phase
[0250] In this experiment response-surface methodology (J. ind.
Microbiol. Biotechnol. 37:195-204 (2010)) was used to determine
optimal values for three parameters, in order to maximize
periplasmic production of a recombinant protein. The three
fermentation parameters investigated were the pH during the growth
phase, the pH during induction and the feed rate during induction.
Values for these three parameters were chosen according to a
Doehlert uniform shell design (Doehlert (Applied Statistics
19:231-239 (1970))). Fifteen fermentations were carried out using
the values described in table 5.
[0251] The fermentations were carried out using strain B2284, this
is a strain of BLR (DE3) cells transformed with a pET26b derivative
containing a sequence coding for a fusion protein between the
signal peptide of FlgI from E. coli and the mature part of CRM197
(this is plasmid pRIT16681 described in FIG. 3 and example 3).
[0252] For each fermentation, the seed culture was thawed to room
temperature and 500 .mu.l was used to inoculate a 2 litre
Erlenmeyer flask containing 400 ml of preculture medium (adapted
from Zabriskie et al. (J. Ind. Microbiol. 2:87-95 (1987))).
[0253] The inoculated flask was then incubated at 37.degree. C.
(.+-.1.degree. C.) and 200 rpm. The pre-culture was stopped when
the optical density at 650 nm (OD.sub.650 nm) reached around 2.5,
(around 6 h of incubation). The pre-culture was used to inoculate
medium in a fermenter as soon as the culture was stopped (adapted
from Zabriskie et al. (J. Ind. Microbiol. 2:87-95 (1987)).
[0254] A 20 litre fermenter (Biolafitte) was used. Nine litres of
batch phase medium were aseptically transferred into the fermenter.
The pH of the medium was readjusted to the target value (Table 5)
with base addition. 3 ml of undiluted irradiated antifoam (SAG 471)
was also added to the fermenter. The temperature (28.degree. C.),
head pressure (0.5 bar), aeration rate (20 litres sparged air per
minute) and initial agitation speed (300 rpm) were then set prior
to inoculation. The level of dissolved oxygen (DO) in these
conditions was 100%. The head pressure and aeration rate were
maintained at a constant level during the fermentation.
[0255] Inoculation was achieved by the addition of 15-20 ml of
pre-culture.
[0256] During batch phase (0-15 h), the temperature was maintained
at 28.degree. C. The level of dissolved oxygen was set at 20% and
regulated by increasing stirring when the DO fell below 20% During
the fed-batch phase (15-46 h), the pH was maintained according to
one of the conditions described in table 5 by addition of base. The
temperature was regulated at 28.degree. C. The stirring rate was
maintained at a constant setpoint (maximum 800 rpm), and the DO
level was maintained at 20% by automatic addition of concentrated
feed solution (adapted from Zabriskie et al. (J. Ind. Microbiol.
2:87-95 (1987)) when the DO increased above 20%.
[0257] When the culture reached an OD.sub.650 nm around 90, the pH
setpoint was modified according to one of the conditions described
in table 5 by base or acid addition and the temperature was
decreased to 23.degree. C. Once these conditions were achieved IPTG
was added to a final concentration of 1 mM. The pH and temperature
were maintained during the whole induction phase (24 h). A constant
substrate feed rate was used during the whole induction phase,
according to one of the conditions described in table 5. The DO
level was maintained at 20% by controlling the stirring rate.
[0258] At the end of the induction phase, cell paste was collected
by centrifugation (typically 6,500.times.g, 4.degree. C. for 1 h),
and stored at -20.degree. C.
[0259] Periplasmic extraction was performed by osmotic shock using
a procedure adapted from Chen et al. (Biochem. Eng. J. 19:211-215
(2004)). CRM197 content in the periplasmic and cytoplasmic
fractions were assayed by Elisa (table 6).
TABLE-US-00005 TABLE 5 Feed rate pH during OD650 nm Culture before
during induction at end of No. induction induction (ml/min)
induction fermentation CDT337 7.0 7.8 1.10 93.0 104.0 CDT338 7.0
7.8 0.28 93.0 102.4 CDT341 7.0 8.7 0.89 94.4 40.0 CDT342 7.0 6.9
0.48 89.2 98.0 CDT344 7.0 6.9 0.89 90.0 89.0 CDT345 7.0 8.7 0.48
92.8 42.0 CDT348 7.0 7.8 0.69 89.2 97.6 CDT349 7.0 7.8 0.69 96.0
109.0 CDT360 7.4 8.1 0.89 88.4 98.8 CDT351 6.6 7.5 0.48 89.2 99.0
CDT354 6.6 7.5 0.89 89.0 93.6 CDT355 6.6 8.4 0.69 87.0 40.0 CDT357
7.4 7.1 0.48 84.8 88.8 CDT358 7.4 7.2 0.69 84.0 86.4 CDT358 7.0 7.8
0.69 94.8 108.0
TABLE-US-00006 TABLE 6 Feed rate CRM197 during (mg/L by Elisa) pH
before pH during induction Cyto- Culture no. induction induction
(ml/min) Periplasmic plasmic CDT337 7.0 7.8 1.10 1500 422 CDT338
7.0 7.8 0.28 921 357 CDT341 7.0 8.7 0.89 13 11 CDT342 7.0 6.9 0.48
1058 341 CDT344 7.0 6.9 0.89 822 275 CDT345 7.0 8.7 0.48 10 20
CDT348 7.0 7.8 0.69 1166 558 CDT349 7.0 7.8 0.69 889 652 CDT360 7.4
8.1 0.89 77 50 CDT351 6.6 7.5 0.48 1533 427 CDT354 6.6 7.5 0.89 803
595 CDT355 6.6 8.4 0.69 20 32 CDT357 7.4 7.1 0.48 54 29 CDT358 7.4
7.2 0.69 681 310 CDT359 7.0 7.8 0.69 1523 685
[0260] Based on the results from the 15 fermentations, the NEMROD-W
software (LPRAI, Marseille, France) was used to model the
production of CRM197 in the periplasmic and cytoplasmic
fractions.
[0261] As shown in FIG. 10, the production of periplasmic CRM197 is
maximal at low feed rates during induction (FIG. 10a), while the
accumulation of CRM197 inside the cell is maximal at higher feed
rates (FIG. 10b). The difference in feed rate optima for the
production of periplasmic or cell-associated CRM197 allows for
defining conditions that selectively improve the production of
periplasmic CRM197. A pH increase at induction is also required for
efficient production of periplasmic CRM197 (FIG. 10a).
[0262] Table 7 below describes the optimum pH during growth, the
optimum pH during induction and the optimum feed rate, as obtained
using the NEMROD-W software.
TABLE-US-00007 TABLE 8 Parameter Value pH during growth 6.8 pH
during induction 7.5 Feed rate during induction 0.330 ml
min.sup.-1
[0263] Optimising the pH and feed rate conditions led to optimal
secretion of CRM97 into the periplasm.
Sequence CWU 1
1
45172DNAArtificial SequencePhtE signal sequence 1atgaaattta
gtaaaaaata tatagcagct ggatcagctg ttatcgtatc cttgagtcta 60tgtgcctatg
ca 72224PRTArtificial SequencePhtE signal sequence 2Met Lys Phe Ser
Lys Lys Tyr Ile Ala Ala Gly Ser Ala Val Ile Val1 5 10 15 Ser Leu
Ser Leu Cys Ala Tyr Ala 20 375DNAArtificial SequenceSipA signal
sequence 3atgaaaatga ataaaaaggt actattgaca tcgacaatgg cagcttcgct
attatcagtc 60gcaagtgttc aagca 75425PRTArtificial SequenceSipA
signal sequence 4Met Lys Met Asn Lys Lys Val Leu Leu Thr Ser Thr
Met Ala Ala Ser1 5 10 15 Leu Leu Ser Val Ala Ser Val Gln Ala 20 25
563DNAArtificial SequenceOmpA signal sequence 5atgaaaaaga
cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60gcc
63621PRTArtificial SequenceOmpA signal sequence 6Met Lys Lys Thr
Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15 Thr Val
Ala Gln Ala 20 757DNAArtificial SequenceNspA signal sequence
7atgaaaaaag cacttgccac actgattgcc ctcgctctcc cggccgccgc actggcg
57819PRTArtificial SequenceNspA signal sequence 8Met Lys Lys Ala
Leu Ala Thr Leu Ile Ala Leu Ala Leu Pro Ala Ala1 5 10 15 Ala Leu
Ala954DNAArtificial SequenceTorT signal sequence 9atgcgcgtac
tgctattttt acttctttcc cttttcatgt tgccggcatt ttcg
541018PRTArtificial SequenceTorT signal sequence 10Met Arg Val Leu
Leu Phe Leu Leu Leu Ser Leu Phe Met Leu Pro Ala1 5 10 15 Phe
Ser1169DNAArtificial SequenceSfmC signal sequence 11atgatgacta
aaataaagtt attgatgctc attatatttt atttaatcat ttcggccagc 60gcccatgct
691223PRTArtificial SequenceSfmC signal sequence 12Met Met Thr Lys
Ile Lys Leu Leu Met Leu Ile Ile Phe Tyr Leu Ile1 5 10 15 Ile Ser
Ala Ser Ala His Ala 20 1375DNAArtificial SequenceFocC signal
sequence 13atgatgaagc acatgcgtat atgggccgtt ctggcatcat ttttagtctt
tttttatatt 60ccgcagagct atgcc 751425PRTArtificial SequenceFocC
signal sequence 14Met Met Lys His Met Arg Ile Trp Ala Val Leu Ala
Ser Phe Leu Val1 5 10 15 Phe Phe Tyr Ile Pro Gln Ser Tyr Ala 20 25
1554DNAArtificial SequenceCcmH signal sequence 15atgaggtttt
tattgggcgt gctgatgctg atgatctccg gctcagcgct ggcg
541618PRTArtificial SequenceCcmH signal sequence 16Met Arg Phe Leu
Leu Gly Val Leu Met Leu Met Ile Ser Gly Ser Ala1 5 10 15 Leu
Ala1769DNAArtificial SequenceYraI signal sequence 17atgtcaaaac
gaacattcgc ggtgatatta accttgttgt gtagcttctg tattggccag 60gcgcttgca
691823PRTArtificial SequenceYraI signal sequence 18Met Ser Lys Arg
Thr Phe Ala Val Ile Leu Thr Leu Leu Cys Ser Phe1 5 10 15 Cys Ile
Gly Gln Ala Leu Ala 20 1966DNAArtificial SequenceTolB signal
sequence 19atgatgaagc aggcattacg agtagcattt ggttttctca tactgtgggc
atcagttctg 60catgct 662022PRTArtificial SequenceTolB signal
sequence 20Met Met Lys Gln Ala Leu Arg Val Ala Phe Gly Phe Leu Ile
Leu Trp1 5 10 15 Ala Ser Val Leu His Ala 20 2166DNAArtificial
SequenceNikA signal sequence 21atgctctcca cactccgccg cactctattt
gcgctgctgg cttgtgcgtc ttttatcgtc 60catgcc 662222PRTArtificial
SequenceNikA signal sequence 22Met Leu Ser Thr Leu Arg Arg Thr Leu
Phe Ala Leu Leu Ala Cys Ala1 5 10 15 Ser Phe Ile Val His Ala 20
2357DNAArtificial SequenceFlgI signal sequence 23atgattaaat
ttctctctgc attaattctt ctactggtca cgacggcggc tcaggct
572419PRTArtificial SequenceFlgI signal sequence 24Met Ile Lys Phe
Leu Ser Ala Leu Ile Leu Leu Leu Val Thr Thr Ala1 5 10 15 Ala Gln
Ala2557DNAArtificial SequenceDsbA signal sequence 25atgaaaaaga
tttggctggc gctggctggt ttagttttag cgtttagcgc atcggcg
572619PRTArtificial SequenceDsbA signal sequence 26Met Lys Lys Ile
Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser1 5 10 15 Ala Ser
Ala2739DNAArtificial SequencePrimer 27gcgcggcata tgggtgcgga
tgatgtggtg gatagcagc 392833DNAArtificial SequencePrimer
28gcggagctcg agttattagc ttttgatttc gaa 332990DNAArtificial
SequencePrimer 29ggagcgcata tgattaaatt tctctctgca ttaattcttc
tactggtcac gacggcggct 60caggctggtg cggatgatgt ggtggatagc
903024DNAArtificial SequencePrimer 30cacgccgcat agttcgcacc cgca
24311605DNAC. diphtheriae mutant CRM197 31ggtgcggatg atgtggtgga
tagcagcaaa tcttttgtga tggaaaactt tagcagctat 60catggcacca aaccgggcta
tgtggatagc attcagaaag gcatccagaa accgaaaagc 120ggcacccagg
gcaactatga tgatgattgg aaagaatttt atagcaccga taacaaatat
180gatgcggcgg gttatagcgt ggataacgaa aatccgctgt ctggcaaagc
gggcggtgtg 240gtgaaagtga cctatccggg cctgaccaaa gtgctggccc
tgaaagtgga taacgcggaa 300accatcaaaa aagaactggg cctgagcctg
accgaaccgc tgatggaaca ggtgggcacc 360gaagaattta ttaaacgctt
tggcgatggc gcgagccgtg tggttctgag cctgccgttt 420gcggaaggca
gcagcagcgt ggaatatatt aacaactggg aacaggcgaa agccctgagc
480gtggaactgg aaattaactt tgaaacccgt ggcaaacgtg gccaggatgc
gatgtatgaa 540tacatggcgc aggcgtgcgc gggcaatcgt gtgcgtcgta
gcgtgggcag cagcctgagc 600tgcattaacc tggattggga cgtcattcgt
gataaaacca aaaccaaaat cgaaagcctg 660aaagaacatg gcccgatcaa
aaacaaaatg agcgaaagcc cgaacaaaac cgtgagcgaa 720gaaaaagcga
aacagtatct ggaagaattt catcagaccg cgctggaaca tccggaactg
780agcgaactga aaaccgtgac cggcaccaat ccggtgtttg cgggtgcgaa
ctatgcggcg 840tgggcggtga atgtggcgca ggtgattgat agcgaaaccg
cggataacct ggaaaaaacc 900accgcggccc tgagcattct gccgggcatt
ggcagcgtga tgggcattgc ggatggcgcg 960gtgcatcata acaccgaaga
aattgtggcg cagagcattg ccctgagcag cctgatggtg 1020gcgcaggcga
ttccgctggt tggcgaactg gtggatattg gctttgcggc gtacaacttt
1080gtggaaagca tcatcaacct gtttcaggtg gtgcataaca gctataaccg
tccggcgtat 1140tctccgggtc ataaaaccca gccgtttctg catgatggct
atgcggtgag ctggaacacc 1200gtggaagata gcattattcg taccggcttt
cagggcgaaa gcggccatga tattaaaatt 1260accgcggaaa acaccccgct
gccgattgcg ggtgttctgc tgccgaccat tccgggcaaa 1320ctggatgtga
acaaaagcaa aacccatatt agcgtgaacg gtcgtaaaat tcgtatgcgt
1380tgccgtgcga ttgatggcga tgtgaccttt tgccgtccga aaagcccggt
gtatgtgggc 1440aacggcgtgc acgcgaacct gcatgtggcg tttcatcgta
gcagcagcga aaaaatccat 1500agcaacgaaa ttagcagcga tagcattggc
gtgctgggct atcagaaaac cgtggaccat 1560accaaagtga actctaaact
gagcctgttc ttcgaaatca aaagc 160532535PRTC. diphtheriae mutant
CRM197 32Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met
Glu Asn1 5 10 15 Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val
Asp Ser Ile Gln 20 25 30 Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr
Gln Gly Asn Tyr Asp Asp 35 40 45 Asp Trp Lys Glu Phe Tyr Ser Thr
Asp Asn Lys Tyr Asp Ala Ala Gly 50 55 60 Tyr Ser Val Asp Asn Glu
Asn Pro Leu Ser Gly Lys Ala Gly Gly Val65 70 75 80 Val Lys Val Thr
Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val 85 90 95 Asp Asn
Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu 100 105 110
Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly 115
120 125 Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly
Ser 130 135 140 Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys
Ala Leu Ser145 150 155 160 Val Glu Leu Glu Ile Asn Phe Glu Thr Arg
Gly Lys Arg Gly Gln Asp 165 170 175 Ala Met Tyr Glu Tyr Met Ala Gln
Ala Cys Ala Gly Asn Arg Val Arg 180 185 190 Arg Ser Val Gly Ser Ser
Leu Ser Cys Ile Asn Leu Asp Trp Asp Val 195 200 205 Ile Arg Asp Lys
Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly 210 215 220 Pro Ile
Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu225 230 235
240 Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu
245 250 255 His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn
Pro Val 260 265 270 Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn
Val Ala Gln Val 275 280 285 Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu
Lys Thr Thr Ala Ala Leu 290 295 300 Ser Ile Leu Pro Gly Ile Gly Ser
Val Met Gly Ile Ala Asp Gly Ala305 310 315 320 Val His His Asn Thr
Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser 325 330 335 Ser Leu Met
Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp 340 345 350 Ile
Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe 355 360
365 Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His
370 375 380 Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp
Asn Thr385 390 395 400 Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln
Gly Glu Ser Gly His 405 410 415 Asp Ile Lys Ile Thr Ala Glu Asn Thr
Pro Leu Pro Ile Ala Gly Val 420 425 430 Leu Leu Pro Thr Ile Pro Gly
Lys Leu Asp Val Asn Lys Ser Lys Thr 435 440 445 His Ile Ser Val Asn
Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile 450 455 460 Asp Gly Asp
Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly465 470 475 480
Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser 485
490 495 Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val
Leu 500 505 510 Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser
Lys Leu Ser 515 520 525 Leu Phe Phe Glu Ile Lys Ser 530 535
3354PRTArtificial SequencePhtE signal sequence and the first 30
amino acids of CRM197 33Met Lys Phe Ser Lys Lys Tyr Ile Ala Ala Gly
Ser Ala Val Ile Val1 5 10 15 Ser Leu Ser Leu Cys Ala Tyr Ala Gly
Ala Asp Asp Val Val Asp Ser 20 25 30 Ser Lys Ser Phe Val Met Glu
Asn Phe Ser Ser Tyr His Gly Thr Lys 35 40 45 Pro Gly Tyr Val Asp
Ser 50 3455PRTArtificial SequenceSipA signal sequence and the first
30 amino acids of CRM197 34Met Lys Met Asn Lys Lys Val Leu Leu Thr
Ser Thr Met Ala Ala Ser1 5 10 15 Leu Leu Ser Val Ala Ser Val Gln
Ala Gly Ala Asp Asp Val Val Asp 20 25 30 Ser Ser Lys Ser Phe Val
Met Glu Asn Phe Ser Ser Tyr His Gly Thr 35 40 45 Lys Pro Gly Tyr
Val Asp Ser 50 55 3551PRTArtificial SequenceOmpA signal sequence
and the first 30 amino acids of CRM197 35Met Lys Lys Thr Ala Ile
Ala Ile Ala Val Ala Leu Ala Gly Phe Ala1 5 10 15 Thr Val Ala Gln
Ala Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser 20 25 30 Phe Val
Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr 35 40 45
Val Asp Ser 50 3649PRTArtificial SequenceNspA signal sequence and
the first 30 amino acids of CRM197 36Met Lys Lys Ala Leu Ala Thr
Leu Ile Ala Leu Ala Leu Pro Ala Ala1 5 10 15 Ala Leu Ala Gly Ala
Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val 20 25 30 Met Glu Asn
Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp 35 40 45 Ser
3748PRTArtificial SequenceTorT signal sequence and the first 30
amino acids of CRM197 37Met Arg Val Leu Leu Phe Leu Leu Leu Ser Leu
Phe Met Leu Pro Ala1 5 10 15 Phe Ser Gly Ala Asp Asp Val Val Asp
Ser Ser Lys Ser Phe Val Met 20 25 30 Glu Asn Phe Ser Ser Tyr His
Gly Thr Lys Pro Gly Tyr Val Asp Ser 35 40 45 3853PRTArtificial
SequenceSfmC signal sequence and the first 30 amino acids of CRM197
38Met Met Thr Lys Ile Lys Leu Leu Met Leu Ile Ile Phe Tyr Leu Ile1
5 10 15 Ile Ser Ala Ser Ala His Ala Gly Ala Asp Asp Val Val Asp Ser
Ser 20 25 30 Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly
Thr Lys Pro 35 40 45 Gly Tyr Val Asp Ser 50 3955PRTArtificial
SequenceFocC signal sequence and the first 30 amino acids of CRM197
39Met Met Lys His Met Arg Ile Trp Ala Val Leu Ala Ser Phe Leu Val1
5 10 15 Phe Phe Tyr Ile Pro Gln Ser Tyr Ala Gly Ala Asp Asp Val Val
Asp 20 25 30 Ser Ser Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr
His Gly Thr 35 40 45 Lys Pro Gly Tyr Val Asp Ser 50 55
4048PRTArtificial SequenceCcmH signal sequence and the first 30
amino acids of CRM197 40Met Arg Phe Leu Leu Gly Val Leu Met Leu Met
Ile Ser Gly Ser Ala1 5 10 15 Leu Ala Gly Ala Asp Asp Val Val Asp
Ser Ser Lys Ser Phe Val Met 20 25 30 Glu Asn Phe Ser Ser Tyr His
Gly Thr Lys Pro Gly Tyr Val Asp Ser 35 40 45 4153PRTArtificial
SequenceYraI signal sequence and the first 30 amino acids of CRM197
41Met Ser Lys Arg Thr Phe Ala Val Ile Leu Thr Leu Leu Cys Ser Phe1
5 10 15 Cys Ile Gly Gln Ala Leu Ala Gly Ala Asp Asp Val Val Asp Ser
Ser 20 25 30 Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly
Thr Lys Pro 35 40 45 Gly Tyr Val Asp Ser 50 4252PRTArtificial
SequenceTolB signal sequence and the first 30 amino acids of CRM197
42Met Met Lys Gln Ala Leu Arg Val Ala Phe Gly Phe Leu Ile Leu Trp1
5 10 15 Ala Ser Val Leu His Ala Gly Ala Asp Asp Val Val Asp Ser Ser
Lys 20 25 30 Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr
Lys Pro Gly 35 40 45 Tyr Val Asp Ser 50 4352PRTArtificial
SequenceNikA signal sequence and the first 30 amino acids of CRM197
43Met Leu Ser Thr Leu Arg Arg Thr Leu Phe Ala Leu Leu Ala Cys Ala1
5 10 15 Ser Phe Ile Val His Ala Gly Ala Asp Asp Val Val Asp Ser Ser
Lys 20 25 30 Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr
Lys Pro Gly 35 40 45 Tyr Val Asp Ser 50 4449PRTArtificial
SequenceFlgI signal sequence and the first 30 amino acids of CRM197
44Met Ile Lys Phe Leu Ser Ala Leu Ile Leu Leu Leu Val Thr Thr Ala1
5 10 15 Ala Gln Ala Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe
Val 20 25 30 Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly
Tyr Val Asp 35 40 45 Ser 4549PRTArtificial SequenceDsbA signal
sequence and the first 30 amino acids of CRM197 45Met Lys Lys Ile
Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser1 5 10 15 Ala Ser
Ala Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val 20 25 30
Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp
35 40 45 Ser
* * * * *